Cell cycle analysis by DNA flow cytofluorimetry and autoradiography has been utilized to investigate the effects of 3-methoxybenzamide (MBA), a potent inhibitor of ADP-ribosylation reactions, on cell cycle progression in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated C3H10T1/2 cells. Following a dose of 6.8 microM MNNG, the presence of MBA resulted in an increased length of S phase from approximately 6.5 h to 10 h and in an accumulation of cells in G2 with a mitosis delay of 12 h. Progression to the next S phase occurred 5-10 times more slowly and the cells ultimately accumulated in G2. Increasing the dose of MNNG resulted in a complete block in cell division in the absence of ADP-ribosylation. These results suggest that ADP-ribosylation reactions, which do not seem to be necessary for DNA excision repair in nondividing cells, are essential for coordinating the events of DNA excision repair with DNA replication and events related to progression through the cell cycle.