The leader of Southern Sudan, Salva Kiir, has urged people to sign up "en masse" for the referendum on the region's independence due in January.
He was speaking to a crowd of hundreds in the southern capital, Juba, as voter registration began after several delays.
The referendum is part of a 2005 deal to end decades of conflict between north and south Sudan.
On Sunday, both sides agreed on ways to ease tensions ahead of the vote.
They agreed to demarcate the north-south border, while allowing northern nomads to graze in the south.
The framework also includes commitments to allow Sudanese citizens the right to live in either north or south Sudan.
Mr Kiir, a former rebel leader who leads the south's semi-autonomous government, was among the first to sign up to vote.
"A referendum happens only once. People must come out en masse otherwise it would mean people fought and died for nothing," he said.
Campaigners with loudspeakers roamed Juba's streets, urging residents to register for the referendum, in which analysts expect southerners to vote to split Africa's biggest nation into two.
The mainly Muslim, Arabic-speaking north has fought the south, where most people are Christian or follow traditional religions, for most of Sudan's independent history.
Southerners are also able to register in Khartoum and several neighbouring countries but Mr Kiir's SPLM government wants them to register in the south.
Some fear that President Omar al-Bashir's national government will somehow prevent the oil-rich south from seceding.
Southern Information Minister Bernaba Benjamin told BBC World Service that the international community needed to keep a close eye on Sudan as the referendum approaches.
"There has been a lot of dragging of feet on the side of our partners in Khartoum with regards to the registration of voters. We fear that rigging of any kind will not help the process of peace in the Sudan".
But the BBC's James Copnall in Khartoum says Sunday's deal has helped to ease some of those fears.
"In the event of secession, this will be the longest inter-state border in Africa, hosting in its immediate vicinity on both sides a significant proportion of Sudan's population," said a statement from the Africa Union, which brokered the deal.
"The parties have committed themselves to maintaining a 'soft border', which will permit unhindered economic and social activity and interaction, which will be essential for economic prosperity and harmony between the north and the south."
Under the new framework, the disputed border area of Abyei will be addressed by direct talks between the presidents of Sudan and the semi-autonomous south, the statement said.
Registration has started late, in part because of a number of political disagreements between north and south - such as demarcating their common border.
An estimated five million people are eligible to vote, and officials now have a shortened period of 17 days in which to register them ahead of the referendum due on 9 January 2011.
Getting material to all the registration centres, in one of the least developed places on earth, has been a real challenge, our correspondent says.
The United Nations has warned logistical problems are likely in some places on the first day.
Denis Kadima, director of the UN's Integrated Referendum and Electoral Division, said most of the materials should be in place, despite the huge logistical challenges, particularly in the south.
But in the Khartoum suburb of Gereif, things did not begin well, says our correspondent, who is at at a registration centre there.
The location of the registration centre was changed at the last minute, leaving domestic observers waiting outside the locked gate of the old venue.
At the new place, posters encouraged every southerner to register, but initially there were more posters than people, our correspondent says.