The biological activity of retinoic acid (RA) was examined in human hepatoma Hep3B cells. Under serum-deprived conditions, RA induced S/M-phase elevation and mitotic index increase within 24 h, followed by apoptosis. This RA-induced apoptosis was accompanied by p53-independent up-regulation of endogenous p21 CIPI/Waf1 and Bax proteins, as well as activation of p34 cdc2 kinase, and increase of Rb2 protein level and phosphorylation pattern. In addition, RA had no effect on the levels of Bcl-X L ; Bcl-X S ; cyclins A, B, D1, D3, or E; or Rb1 expression but markedly down-modulated Cdk2 kinase activity and reduced Cdk4 expression. RA also slightly delayed p27 Kip1 expression. Olomoucine, a potent p34 cdc2 and Cdk2 inhibitor, effectively blocked RA-mediated p34 cdc2 kinase activation and prevented RA-induced apoptosis. Furthermore, antisense oligonucleotide complementary to p21 CIP2/Waf1 and p34 cdc2 mRNA significantly rescued RA-induced apoptosis. Our data indicate that p21 CIP2/Waf1 overexpression may not be the only regulatory factor necessary for RA-induced apoptosis in human hepatoma Hep3B cells. RA treatment leads to Rb2 hyperphosphorylation, and p34 cdc2 kinase activation is coincident with an aberrant mitotic progression, followed by appearance of abnormal nucleus. This aberrant cell cycle progression appeared requisite for RA-induced cell death. These findings suggest that inappropriate regulation of the cell cycle regulators p21 CIP2/Waf1 and p34 cdc2 is coupled with induction of Bax and involved in cell death with apoptosis when Hep3B cells are exposed to RA.