It has been shown that 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3) and dietary Ca modulate renal metabolism of 25-hydroxyvitamin D3 (25-OH-D3) to 1,25-(OH)2-D3 and 24,25-dihydroxyvitamin D3 (24,25-(OH)2-D3) in the rat. However, it is not known if 1,25-(OH)2-D3 and Ca act directly on the kidney to modulate 25-OH-D3 metabolism or indirectly through other mechanisms, such as the modulation of parathyroid hormone secretion. Therefore, we have used isolated renal cortical slices from the rat to study the effect of 1,25-(OH)2-D3 and Ca in vitro on renal 25-OH-D3 metabolism. Incubation of renal slices from rats fed a vitamin D-deficient, low-Ca diet with 50 nM 1,25-(OH)2-D3 for 3 h resulted in a significant decrease in 1,25-(OH)2-D3 production and a significant increase in 24,25-(OH)2-D3 production. Increasing media Ca concentration from 0.5 to 2.5 mM resulted in a significant decrease in 1,25-(OH)2-D3 production but no change in 24,25-(OH)2-D3 production. The inhibitory effect of 1,25-(OH)2-D3 was blocked by cycloheximide, but the inhibitory effect of Ca was not blocked by cycloheximide. Renal 1,25-(OH)2-D3 production was inhibited to a greater extent by incubation with 1,25-(OH)2-D3 and Ca together than by incubation with 1,25-(OH)2-D3 and Ca separately. These studies indicate that 1,25-(OH)2-D3 and Ca act directly on the kidney to modulate renal 25-OH-D3 metabolism. They also suggest that the mechanism of modulation is different for each agent.