1Alpha,25-dihydroxyvitamin D3 (1,25 D), the most active metabolite of vitamin D3, exerts antiproliferative and prodifferentiating effects on some human prostate cancer cell lines. We previously reported an inverse relationship between functional vitamin D receptor (VDR) levels and antiproliferative response to 1,25 D in two human prostate cancer cell lines, LNCaP and ALVA 31. Although LNCaP cells are far more sensitive to growth inhibition by 1,25 D than ALVA 31 cells, LNCaP express approximately half the number of VDR as ALVA 31. Two other human prostate cancer cell lines studied, PC3 and DU145, express lower levels of functional VDR and are relatively insensitive to growth inhibition by 1,25 D. In this report, we investigated potential mechanisms of the variable antiproliferative activity of 1,25 D. In PC3 cells stably expressing VDR [PC3(VDR)] at levels comparable to LNCaP, 1,25 D treatment resulted in only moderate growth inhibition. These results further support the contention that VDR expression, although required, is not sufficient for maximal growth suppression by 1,25 D, as is exhibited by LNCaP cells. We did not detect 1,25 D-mediated DNA fragmentation after 4 days of 1,25 D treatment in either LNCaP or ALVA 31 cells. This result suggests that variability in 1,25 D sensitivity does not derive from differences in the capacity of these cells to undergo apoptosis in response to 1,25 D. Flow cytometry of propidium iodine-stained cells revealed that 48 h 1,25 D treatment of LNCaP cells resulted in a 2-fold decrease of cells in G2/M plus S phases and accumulation of LNCaP cells in the G1/G0 phase. This effect persisted for 72 h after 1,25 D removal. In contrast, 1,25 D did not significantly alter the cell cycle distribution of ALVA 31 or PC3(VDR) cells. Consistent with accumulation of cells in G1/G0, 1,25 D treatment of LNCaP cells resulted in decreased retinoblastoma protein phosphorylation, repressed E2F transcriptional activity, increased levels of the cyclin-dependent kinase (CDK) inhibitor p21(WAF1, CIP1), and decreased CDK2 activity. However, p21 messenger RNA levels were not altered, suggesting translational or posttranslational regulation of p21 by 1,25 D. In contrast, p21 was not detected in ALVA 31 or PC3(VDR) and was not induced by 1,25 D, consistent with the failure of 1,25 D to influence cell cycle distribution in these cells. These results suggest that variability in sensitivity to the antiproliferative effects of 1,25 D among prostate cancer cells is dependent, at least in part, on the integrity of the retinoblastoma pathway and in particular on p21 expression and 1,25 D regulation of CDK2 activity.