We have previously shown that replication in vitro of plasmids containing the Simian virus 40 (SV40) origin of replication is reduced when an extract of irradiated cells is used (Wang et al., Radiat. Res. 142, 169-175, 1995). We proposed that the observed reduction in the overall replication activity is due to a reduction in the efficiency of initiation events, and that it is caused by the induction or activation by ionizing radiation of a factor(s) that inhibits DNA replication in trans. Here, we extend these studies and provide evidence that the reduced replication activity of an extract prepared from irradiated cells is not the result of a nonspecific inactivation of proteins or of an increase in the requirement for SV40 large tumor antigen (TAg), the only noncellular protein required for in vitro DNA replication. Mixing experiments demonstrate the presence of a dominant inhibitory activity(ies) in the extract of irradiated cells that efficiently stalls replication in reactions assembled using extract of nonirradiated cells. The inhibitory activity is a stable, nondialyzable molecule. Studies of kinetics suggest that the inhibitory activity(ies) affects the initiation steps of DNA replication and acts, at least partly, by modifying TAg, the key initiation protein of SV40 ori DNA replication. It is likely that the same inhibitory activity(ies) regulates cellular DNA replication by modifying the cellular homologues of TAg. Purification and characterization of this inhibitory activity(ies) will contribute to our understanding of the mechanism developed by the cell to regulate DNA replication after exposure to ionizing radiation and will define a checkpoint operating in S phase. Genetic evidence for a checkpoint in S phase distinct from the checkpoints operating in G1 and G2 phase has been reported in yeast.