The region at and near the simian virus 40 (SV40) DNA replication origin contains a series of palindromes, a 17-base pair (bp) A + T-rich sequence, three copies of a 21-bp repeat, and two copies of a 72-bp repeat. We have constructed a series of recombinant plasmids containing sequential deletions at the region of SV40 DNA replication origin starting from the end near the repeats. These deletions were introduced by using in vitro and in vivo techniques. The relative replication efficiency of these recombinant plasmids were directly assayed in COS-1 monkey kidney cells capable of providing the tumor antigen necessary for the replication of these molecules. Recombinants lacking both copies of the 72-bp repeat did not exhibit any reduction in replication efficiency. Recombinants lacking the 21-bp repeats showed decreased replication efficiency; the reduction in replication efficiency was proportional to the number of copies of the 21-bp repeat deleted in these recombinants. A recombinant retaining the palindromes at the region of SV40 DNA replication but lacking the A + T-rich sequence and the repeats failed to replicate. Based on these results, the SV40 DNA replication origin is subdivided into two regions, and their boundaries are defined. One of these two regions is a core region containing the 17-bp, 15-bp, and 27-bp palindromes and, quite likely, the 17-bp A + T-rich sequence which are necessary for replication. The other is an auxiliary region that consists of the 21-bp repeats and has a dose-dependent enhancement effect on replication efficiency.