Soluble extracts prepared from monkey cells (COS-1 or BSC-40) infected with simian virus 40 (SV40) catalyze the efficient replication of exogenously added plasmid DNA molecules containing the cloned SV40 origin of replication. Extracts prepared from uninfected monkey cells also support origin-dependent replication in vitro but only in the presence of added SV40 large tumor (T) antigen. Very little DNA synthesis is observed when the cloned viral origin contains a 4-base-pair deletion mutation known to abolish SV40 DNA replication in vivo or when the parental plasmid vector lacking SV40 sequences is employed as template. The in vitro replication reaction proceeds via branched intermediates (theta structures) that resemble in vivo replication intermediates. Replication is sensitive to aphidicolin but relatively resistant to dideoxythymidine triphosphate. The product of the reaction consists of covalently closed circular DNA molecules that contain full-length daughter strands hydrogen bonded to the parental template. These observations support the conclusion that replication in the in vitro system closely resembles SV40 DNA replication in vivo. The system provides a biochemical assay for the replication activity of SV40 T antigen and should also facilitate the purification and functional characterization of cellular proteins involved in DNA replication.