Inhibition of DNA replication by different DNA damaging agents has been investigated in HeLaMR cells and a methylation damage-tolerant variant HeLa5A1. In synchronous HeLaMR and HeLa5A1 cells exposed to N-ethyl-N-nitrosourea or ionizing radiation in mid-G1 phase, DNA synthesis was inhibited in the following S phase. N-methyl-N-nitrosourea-induced replication inhibition in HeLaMR cells was delayed until the second S phase after treatment. In contrast, N-methyl-N-nitrosourea treatment of HeLa5A1 cells affected neither the timing nor the extent of the first or second S phases. Both radiation and chemical treatment inhibited replication of an episomal plasmid and of genomic DNA in unison. Inhibition was observed at levels of DNA damage that did not directly damage the plasmid molecules. Thus, DNA replication inhibition occurs immediately after ionizing radiation or ethylation damage, but methylation damage requires processing through one cell cycle to generate an inhibitory signal. The inhibitory signal appears to act in trans on undamaged DNA. Although methylation-tolerant cells are responsive to inhibition after gamma-irradiation, methylation damage does not produce inhibitory signals to which they respond.