Inhibition of polyamine biosynthesis with alpha-difluoromethylornithine (DFMO) has been shown to inhibit proliferation of breast cancer cells although its mechanism of action has not been fully elucidated. To address this issue, we tested the effects of DFMO on cell cycle variables of MDA-MB-435 human breast cancer cells in culture. We also focused on the possible mediatory role of the mitogen-activated protein kinase (MAPK) pathway on the cell cycle effects of DFMO because this compound has been shown to activate MAPK signaling. We found that DFMO caused a p53-independent increase in p21 and its association with cyclin-dependent kinase (cdk)-2 and decreased cdk-2 protein as well as its phosphorylation on Thr160. In addition, DFMO markedly suppressed the expression of the full-length and low molecular weight forms of cyclin E. These effects of DFMO were reversible with exogenous putrescine, thus indicating that they are specifically mediated through polyamine depletion. Cdk-2 activity was drastically reduced in DFMO-treated breast cancer cells which exhibited a reduction in retinoblastoma (Rb) phosphorylation and protein. As a predictable consequence of these effects, DFMO caused a G1-S block. In addition, DFMO inhibited G2-M transition, most likely as a result of its induction of p21 expression. Inhibition of the MAPK pathway with PD98059 or U0126 blocked the DFMO-induced induction of p21 and the reduction of cdk-2 protein. PD98059 reversed the G2-M block induced by DFMO (probably as a result of suppression of p21) but not the G1-S arrest. MDA-MB-435 cells treated with PD98059 or U0126 in the presence and absence of DFMO exhibited a marked increase in the expression of p27 and its association with cdk-2, a decrease in phosphorylation of cdk-2 on Thr160, and a decrease in cyclin E expression. As predicted, PD98059 treatment reduced cdk-2 activity and Rb phosphorylation while reversing the decrease in Rb protein induced by DFMO. Neither DFMO nor PD98059, either alone or in combination, reduced cdk-4 activity despite a marked induction in p15 expression caused by DFMO. Our results indicate that activation of the MAPK pathway accounts for some of the effects of DFMO on cell cycle events of breast cancer cells. Inhibition of the MAPK pathway, however, does not reverse the cell cycle arrest induced by DFMO because of activation of alternative mechanisms leading to suppression of cdk-2 activity.