Transforming growth factor-beta1 (TGF-beta1) can induce rapid growth arrest and apoptosis in hepatic cells. Its growth suppressive effects appear to be linked to decreased phosphorylation of the protein product of the retinoblastoma gene, pRb. To characterize the role of pRb in apoptosis, we examined endogenous retinoblastoma gene (Rb) expression following treatment with TGF-beta1, okadaic acid, or antisense Rb S-oligonucleotides in cultured primary rat hepatocytes and human hepatoma HuH-7 cells. We also investigated the effects on apoptosis of Rb overexpression following transfection with vectors containing wild-type Rb in HuH-7 cells. Our results indicated that transfection with Rb antisense S-oligonucleotides blocked the expression of pRb in cultured primary hepatocytes and induced apoptosis. Treatment of HuH-7 cells with TGF-beta1 inhibited expression and phosphorylation of pRb, and also induced apoptosis. Furthermore, 93% of viable preapoptotic cells were arrested in the G1 phase of the cell cycle. Incubation with the phosphatase inhibitor okadaic acid maintained pRb in its phosphorylated state, and resulted in significant apoptosis. Overexpression of wild-type Rb inhibited TGF-beta1 induced apoptosis in HuH-7 cells. In contrast, overexpression of transcription factor E2F-1, a known target for the activity of pRb, caused significant apoptosis. However, coexpression of Rb suppressed E2F-1 induced apoptosis in HuH-7 cells. Our results suggest that inhibition of pRb expression is associated with hepatocyte apoptosis. Furthermore, E2F-1 appears to be a target in the pathway through which pRb modulates the apoptotic threshold in hepatic cells. Finally, the data suggest that these cells exit the cell cycle during the G1 phase before progressing into apoptosis and pRb may be a negative regulator of this process.