Renal production of 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] from 25-hydroxyvitamin D₃ (25OHD₃) is increased by PTH. The complete mechanism by which PTH modulates renal 25OHD₃ metabolism is not known, but there is some evidence that the stimulation of renal cAMP production by PTH may be important. Therefore, we have used forskolin, a direct activator of adenylate cyclase in the intact tissue, to further investigate the role of cAMP in regulating renal 25OHD₃ metabolism. The effect of forskolin on renal 25OHD₃ metabolism and renal adenylate cyclase activity was measured using isolated renal slices from thyroparathyroidectomized rats previously fed a vitamin D-deficient, low calcium diet. Forskolin added to renal slices in vitro for 4 h increased renal 1,25-(OH)₂-D₃ production in a concentration-dependent manner. In separate experiments, forskolin was found to increase tissue cAMP in a concentration-dependent manner when added for 5 min. The concentration of forskolin necessary for half-maximal stimulation of adenylate cyclase was 10 μm, and that needed for halfmaximal stimulation of 1,25-(OH)₂-D₃ production was 1 μm. PTH added to renal slices also increased renal 1,25-(OH)₂-D₃ production, but the effects of PTH and forskolin were not additive. Inclusion of 1,25-(OH)₂-D₃ in the incubation medium blocked the effect of forskolin on 1,25-(OH)₂-D₃ production, but it did not block the effect of forskolin on tissue cAMP content. These studies support the concept that forskolin and PTH modulate renal 25OHD₃ metabolism though a cAMP-dependent pathway. However, this pathway may be further regulated at sites distal to cAMP production by compounds such as 1,25-(OH)₂-D₃. (Endocrinology114: 644, 1984)