Renal vitamin D receptor (VDR) is required for 1,25-dihydroxyvitamin D3-[1,25(OH)2D3]-induced renal reabsorption of calcium and for 1,25(OH)2D3-induced 1,25(OH)2D3 24-hydroxylase. The long-term effect of vitamin D and dietary calcium on the expression of renal VDR was examined in the nonobese diabetic mouse. Vitamin D-deficient and vitamin D-replete mice were maintained on diets containing 0.02%, 0.25%, 0.47%, and 1.20% calcium with or without 50 ng of 1,25(OH)2D3 per day. Vitamin D-replete mice on a 1.20% calcium diet had renal VDR levels of 165 fmol/mg protein. Calcium restriction caused renal VDR levels to decrease to <30 fmol/mg protein in vitamin D-deficient mice and to approximately 80 fmol/mg protein in vitamin D-replete mice. When dietary calcium was present, 50 ng of 1,25(OH)2D3 elevated the VDR levels 2- to 10-fold, depending on vitamin D status and the level of calcium. In the absence of either vitamin D or calcium, the VDR mRNA was expressed at a basal level. 1,25(OH)2D3 supplementation caused relative VDR mRNA to increase 8- to 10-fold in the vitamin D-deficient mouse when dietary calcium was available. This increase was completely absent in the calcium-restricted mice. This in vivo study demonstrates that 1,25(OH)2D3 and calcium are both required for renal VDR mRNA expression above a basal level, furthering our understanding of the complex regulation of renal VDR by 1,25(OH)2D3 and calcium.