1,4-Dichlorobenzene (DCB), a non-DNA-reactive compound, induced hepatocellular carcinomas at 600 mg/kg/day, but not 300 mg/kg/day in male and female B6C3F1 mice in a National Toxicology Program (NTP) bioassay. Cell proliferation studies were performed under conditions of the NTP bioassay to determine the mode of DCB-induced hepatocellular proliferation and whether this proliferative response may be related to the carcinogenic activity of DCB. The percentage of cells in S-phase (labeling index; LI) was measured using immunohistochemical detection of 5-bromo-2'-deoxyuridine. Time-course and dose-response studies revealed a sharp increase in LI 24 h after treatment in female mice and rats, and at 48 h in male mice with no increases in liver-associated plasma enzymes at up to twice the highest bioassay dose. During 13 weeks of DCB administration under bioassay conditions, a statistically significant transient peak of hepatocellular proliferation was observed during week 1 at 600 mg/kg/day, but not at 300 mg/kg/day, in male and female mice. Hepatocellular proliferation was also observed in female rats, which were reported as exhibiting no increased liver tumor incidence when compared to controls in the NTP bioassay. An increase in liver weight as a percentage of body weight compared to controls was observed in high dose male and female mice, and female rats at all time points. No significant elevations in liver-associated plasma enzymes were found at any time point, indicating a lack of overt hepatotoxicity. Histopathological evaluation revealed no evidence of hepatocellular necrosis in all groups. These data indicate an early mitogenic stimulation of cell proliferation, rather than regeneration secondary to cytolethality, in the livers of DCB-treated mice, which correlates with previously observed tumor formation in a dose-dependent manner. The mode by which a chemical induces cell proliferation is an important consideration in mechanistic studies and the risk assessment process. The demonstrated mitogenic activity of DCB raises the possibility that this early proliferative response may be sufficient for liver tumor formation in the B6C3F1 mouse, or that DCB may provide a selective growth advantage to preneoplastic cells in the mouse liver upon long-term treatment. The observed induction of cell proliferation by DCB in the rat in the absence of a tumorigenic response suggests important species differences and complexities in the relationship between cell proliferation and carcinogenesis, and indicates that caution be applied in equating cell proliferation to cancer.