The human single-stranded-DNA binding protein (human SSB) is required for simian virus 40 (SV40) DNA replication in vitro. SV40 large tumor antigen and human SSB can support extensive unwinding of SV40 origin-containing DNA in the presence of ATP and a topoisomerase that relieves positive superhelicity. Although SSBs from viral and prokaryotic sources substituted for human SSB in the DNA-unwinding reaction, they did not substitute in the replication of SV40 DNA. The specificity for human SSB in SV40 DNA replication can be explained, at least in part, by the finding that DNA polymerase alpha was stimulated 10-fold by human SSB but not by other SSBs. Human SSB also stimulated proliferating-cell nuclear antigen-dependent DNA polymerase delta; however, other SSBs stimulated this polymerase as well.