We previously showed that calcitriol (1α,25-dihydroxyvitamin D3) induces clonal mouse epidermal JB6 Cl41.5a cells to synthesize and secrete a nonphosphorylated form of osteopontin (OPN) in a dose-dependent and metabolite-specific manner. To investigate whether OPN expression is transcriptionally regulated by calcitriol in these cells, we first determined the early time course of calcitriol-induced expression of OPN protein and steady state levels of OPN messenger RNA (mRNA). Calcitriol treatment of JB6 Cl41.5a cells for 6 h caused increased secretion of [35S]methionine- labeled OPN, with maximal levels attained after 8 h of treatment. Northern analyses showed that steady state levels of OPN mRNA increase before the synthesis and secretion of OPN protein. Treatment of JB6 Cl41.5a cells with calcitriol and the transcriptional inhibitor actinomycin-D (2-250 ng/ml) indicated that calcitriol-induced accumulation of steady state OPN mRNA and secretion of OPN protein were dose dependently inhibited by actinomycin-D. These data suggest that calcitriol regulates the expression of OPN at the level of transcription. Furthermore, calcitriol increased the steady state level of OPN mRNA in a dose-dependent manner. Calcitriol-mediated increases in OPN expression were also observed in a transfection assay using a construct consisting of a portion of the promoter region of the OPN gene fused to the luciferase reporter gene. In addition, a study using 5,6- dichloro-1-β-D-ribofuranosylbenzimidazole, an adenosine analog that inhibits mRNA synthesis, showed that calcitriol treatment did not significantly increase the stability of OPN mRNA. These findings suggest that calcitriol increases the expression of OPN mRNA and protein by stimulating transcription.