Activation of alpha1B adrenergic receptors (alpha(1B)AR) promotes DNA synthesis in primary cultures of hepatocytes, yet expression of alpha(1B)AR in hepatocytes rapidly declines during proliferative events. HepG2 human hepatoma cells, which do not express alpha(1B)AR, were stably transfected with a rat alpha1B(AR) cDNA (TFG2 cells), in order to study the effects of maintained alpha(1B)AR expression on hepatoma cell proliferation. TFG2 cells had a decreased rate of growth compared to mock transfected HepG2 cells as revealed by a decrease in [3H]thymidine incorporation into DNA. Stimulation of alpha(1B)AR with phenylephrine caused a further large reduction in TFG2 cell growth, whereas no effect on growth was observed in mock transfected cells. Reduced cell growth correlated with increased percentages of cells found in G0/G1 and G2/M phases of the cell cycle. In TFG2 cells, phenylephrine increased p42MAPkinase activity by 1.5- to 2.0-fold for up to 24 h and increased expression of the cyclin dependent kinase inhibitor protein p21Cip1/WAF1. Treatment of TFG2 cells with the specific MEKI inhibitor PD98059, or infection with a -/- MEK1 recombinant adenovirus permitted phenylephrine to increase rather than decrease [3H]thymidine incorporation. In addition, inhibition of MAP kinase signaling by PD98059 or MEK1 -/- blunted the ability of phenylephrine to increase p21Cip1/WAF1 expression. In agreement with a role for increased p21Cip1/WAF1 expression in causing growth arrest, infection of TFG2 cells with a recombinant adenovirus to express antisense p21Cip1/WAF1 mRNA blocked the ability of phenylephrine to increase p21Cip1/WAF1 expression and to inhibit DNA synthesis. Antisense p21Cip1/WAF1 permitted phenylephrine to stimulate DNA synthesis in TFG2 cells, and abrogated growth arrest. These results suggest that transformed hepatocytes may turn off the expression of alpha1B(ARs) in order to prevent the activation of a growth inhibitory pathway. Activation of this inhibitory pathway via alpha1B(AR) appears to be p42MAPkinase and p21Cip1/WAF1 dependent.