1,25-Dihydroxycholecalciferol (1,25-D3) has significant antitumor effects in the murine squamous cell carcinoma (SCC) tumor model in vitro and in vivo. We investigated the basis for this antiproliferative activity and found that, in vitro, 1,25-D3 administration is associated with altered expression of cell cycle regulatory proteins, treatment results in retinoblastoma dephosphorylation, decreased expression of p21(Waf1/Cip1) (p21) mRNA and protein, and increased expression of p27Kip1 (p27) mRNA and protein. Dexamethasone, which acts synergistically with 1,25-D3 to inhibit SCC proliferation, enhanced 1,25-D3-induced down-modulation of p21 without affecting the ability of 1,25-D3 to increase p27 expression. 1,25-D3 did not induce cleavage of poly(ADP-ribose) polymerase. These in vitro data suggest that 1,25-D3 exerts antitumor activity in SCC by perturbing cell cycle progression rather than by inducing apoptosis. In vivo, a 1,25-D3 treatment regimen that results in a decrease in SCC tumor volume is associated with a statistically significant decrease in intratumoral p21 expression. p21 expression is not changed in tumors isolated from control animals or animals treated with a nontherapeutic dose of 1,25-D3. Intratumoral p27 levels were not modulated by 1,25-D3 treatment. Thus, both in vitro and in vivo, 1,25-D3-mediated growth inhibition is associated with p21 down-modulation.