The effects of nicotinamide and structural analogs on DNA synthesis were studied in rat hepatoma (HTC) cells. Inhibitory effects of these compounds were observed on DNA synthesis as judged by the incorporation of [3H]-thymidine into DNA. Evidence for a marked effect on DNA integrity after preincubation with 1 mM methyl methanesulfonate was provided by a fluorometric technique with ethidium bromide. There was only a small or insignificant enhancement of this effect when hepatoma cells were incubated with nicotinamide. At concentrations of 2-20 mM, 3-aminobenzamide was observed to cause greater effects than nicotinamide on DNA synthesis and integrity and on cellular proliferation in HTC cells. Comparison of the effects of nicotinamide and 3-aminobenzamide with those of N'-methylnicotinamide suggested that some of the effects on DNA synthesis may not be mediated through inhibition of poly(ADP-ribose) synthetase. Inhibition of HTC cell proliferation was observed at a concentration of 3-aminobenzamide, 2 mM, which has been reported to be nontoxic for other cell types.