Three plasmids were derived from a common SV40-based parent. A circular plasmid (pYACneoC) contained the SV40 ori and two sets of 50 bp of human telomeric sequences. By differential enzyme digestion, two linear plasmids were generated from the circular form, one (pYACneoL) terminating with, and the other (pYACneoN) free of telomeric sequences. The replicative features of the circular and of both linear plasmids were assayed by transfecting COS-7 cells. At various times after transfection, the low-molecular-weight DNA was extracted, and the fraction of molecules that had replicated was determined by Dpnl digestion. We demonstrate that about half of the circular plasmid molecules replicate, but only during a short time interval immediately following transfection. No replication was observed in the case of the two linear plasmids. However, the function of the SV40 origin is potentially present in the molecules, since circular forms that do replicate can be recovered from both linear plasmids. The extent of replication of circularized pYACneoL is similar to that of pYACneoC, whereas a lower fraction of circularized pYACneoN molecules replicate. These results are discussed in terms of the possible influence of the DNA structure on the viral ori, and of the influence of the host cell functions on viral replication.