Previous analysis of simian virus 40 (SV40) DNA replication revealed a 2-4 fold excess of DNA molecules that were 90 +/- 2% replicated, demonstrating that replication forks accumulate near the termination site. To determine whether replication is arrested at specific DNA sites, forks were located on the SV40 genome by specifically 32P labelling 3' ends of nascent DNA on purified replicating SV40 DNA, isolating the longest 32P-DNA chains, annealing them to SV40 DNA and then digesting them with a restriction endonuclease that cut near the terminatin site. 32P-DNA fragments of several discrete lengths were released, demonstrating that replication forks on native chromosomes were arrested at preferred sites on the DNA. Most forks were arrested when bidirectional DNA replication was 91% completed, and the two forks were separated by about 470 bp of unreplicated DNA centered at the expected termination site. Forks were also arrested at other locations such that the center of the termination region defined by DNA arrest sites varied by +/- 450 bp. Electron microscopic analysis of replicating DNA suggested that such variation may result from asynchronous arrival of some replication forks. Analysis of 5' end-labeled nascent DNA demonstrated that initiation of Okazaki fragments was also promoted at preferred DNA sites (about 100-120 per genome). Thus specific DNA sequences appear to be utilized throughout DNA replication, not just at the origin.