Human keratinocyte is one of the target cells for 1,25(OH)2D3, a biologically active form of vitamin D3. It induces the differentiation and growth inhibition of human keratinocytes. In order to understand the inhibitory mechanism of 1,25(OH)2D3, we examined its effect on cell cycle kinetics and retinoblastoma gene product (pRB), one of tumor suppressor gene products, in normal human keratinocytes. Cell cycle analysis demonstrated that 10(-6) M of 1,25(OH)2D3 induced cell cycle arrest in both G1/G0 (63.4% +/- 1.4 versus 52.7% +/- 1.2 in control, p < 0.05) and G2 + M (21.5% +/- 0.6 versus 10.9% +/- 0.8 in control, P < 0.05) phase. Addition of 10(-6) M of 1,25(OH)2D3 increased dephosphorylated pRB in a time dependent manner from 23% at 0 h to 58% at 48 h. Since the phosphorylation of pRB is supposed to be essential for the progression from G1 to S phase, the inhibition of pRB phosphorylation could be responsible for the G1/G0 growth arrest induced by 1,25(OH)2D3 in normal human keratinocytes.