CYC202 (R-roscovitine) is a potent cyclin-dependent kinase inhibitor, investigated as a potential anti-cancer agent. The knowledge of the action of this pharmacological agent on normal human cells is still limited. In this study, we have explored the effects of the cyclin-dependent kinase inhibitor CYC202 on normal human epidermal keratinocytes. The loss of cell viability induced by this compound was strongly dependent on the rate of keratinocyte proliferation. At slightly cytotoxic doses, CYC202 inhibited the proliferation of subconfluent keratinocytes in a dose-dependent manner, and at higher concentrations induction of early apoptosis was observed, evidenced by caspase-3 activation. The signal transduction pathways in subconfluent keratinocytes were altered, as CYC202 increased the phosphorylation of p38 MAP kinase. The activation of this kinase was confirmed by the increased phosphorylation of p38 MAPK substrate, the small heat shock protein HSP27. Prolonged inhibition of highly proliferative cells with CYC202 for 48 and 72 h altered the expression of epidermal differentiation markers. The use of the selective p38 kinase inhibitor PD169316 demonstrated that involucrin mRNA was upregulated by CYC202 via p38 MAPK pathway. These effects were strongly dependent on cell density and were observed only in highly proliferative keratinocytes. We concluded that CYC202 although highly potent against cancer cells inhibits also the proliferation and induces early apoptotic events in autocrine culture of normal human keratinocytes, activates p38 MAP kinase pathway and alters the expression of the epidermal differentiation markers. These results suggest that despite this potency against tumour cells, CYC202 must be used attentively in the clinical practice.