1,25-(OH)2 vitamin D3 (1,25-(OH)2D3) exerts antiproliferative effects via cell cycle regulation in a variety of tumor cells, including prostate. We have previously shown that in the human prostate cancer cell line LN-CaP, 1,25-(OH)2D3 mediates an increase in cyclin-dependent kinase inhibitor p27Kip1 levels, inhibition of cyclin-dependent kinase 2 (Cdk2) activity, hypophosphorylation of retinoblastoma protein, and accumulation of cells in G1. In this study, we investigated the mechanism whereby 1,25-(OH)2D3 increases p27 levels. 1,25-(OH)2D3 had no effect on p27 mRNA levels or on the regulation of a 3.5-kb fragment of the p27 promoter. The rate of p27 protein synthesis was not affected by 1,25-(OH)2D3 as measured by luciferase activity driven by the 5'- and 3'-untranslated regions of p27 that regulate p27 protein synthesis. Pulse-chase analysis of 35S-labeled p27 revealed an increased p27 protein half-life with 1,25-(OH)2D3 treatment. Because Cdk2-mediated phosphorylation of p27 at Thr187 targets p27 for Skp2-mediated degradation, we examined the phosphorylation status of p27 in 1,25-(OH)2D3-treated cells. 1,25-(OH)2D3 decreased levels of Thr187 phosphorylated p27, consistent with inhibition of Thr187 phosphorylation-dependent p27 degradation. In addition, 1,25-(OH)2D3 reduced Skp2 protein levels in LNCaP cells. Cdk2 is activated in the nucleus by Cdk-activating kinase through Thr160 phosphorylation and by cdc25A phosphatase via Thr14 and Tyr15 dephosphorylation. Interestingly, 1,25-(OH)2D3 decreased nuclear Cdk2 levels as assessed by subcellular fractionation and confocal microscopy. Inhibition of Cdk2 by 1,25-(OH)2D3 may thus involve two mechanisms: 1) reduced nuclear Cdk2 available for cyclin binding and activation and 2) impairment of cyclin E-Cdk2-dependent p27 degradation through cytoplasmic mislocalization of Cdk2. These data suggest that Cdk2 mislocalization is central to the antiproliferative effects of 1,25-(OH)2D3.