SV40 (Simian Virus 40) DNA was pulse-labeled with [(3)H]thymidine in infected monkey cells, and the distribution of label within newly completed molecules was determined by analysis of specific fragments produced by restriction endonuclease from Hemophilus influenzae. From these data, an order of synthesis or temporal order of the fragments was deduced. Comparison of the temporal order with the physical order of the fragments in the SV40 DNA molecule indicates a correspondence in these orders for two separate groups of fragments. From an analysis of the results, we conclude that SV40 DNA replication begins at a specific site and proceeds bidirectionally, terminating about halfway around the circular molecule from the initiation point.