Details of the molecular mechanisms determining levels of the secosteroid, 1,25-dihydroxyvitamin D(3) (1,25D) remain to be elucidated. The current paradigm for the control of serum 1,25D levels is the tight regulation of renal 25-hydroxyvitamin D-1alpha-hydroxlase (CYP27B1) activity by a number of physiological factors. 1,25D production is also regulated by the cytochrome P450 enzyme, 25-hydroxyvitamin D-24-hydroxylase (CYP24), which through side chain hydroxylation reactions, inactivates 1,25D. We have recently demonstrated that renal CYP27B1 and CYP24 expression contribute equally to regulating serum 1,25D levels. We now describe the contribution of renal Vitamin D receptor (VDR) expression in determining serum 1,25D levels. Serum 1,25D levels were decreased when the dietary calcium intake was increased. We measured mRNA levels for CYP27B1, CYP24 and VDR receptor in kidney RNA extracts from animals fed diets containing different levels of calcium, ranging from 0.05 to 1%. Serum 1,25D levels were negatively correlated with renal CYP24 mRNA levels (R2 = 0.35, P < 0.01) while renal VDR is positively correlated with renal CYP24 mRNA (R2 = 0.80, P < 0.001). However, only renal VDR mRNA remained a significant determinant of renal CYP24 expression when both these variables were included in multiple linear regression analysis (multiple R2 = 0.89, P < 0.001). These findings suggest that kidney CYP24 activity acts in concert with kidney CYP27B1 to control serum 1,25D levels and that serum 1,25D stimulates renal CYP24 expression by acting through the renal VDR.