Kidneys of adult rats were removed and perfused with semisynthetic media with the object of elucidating the separate actions of factors implicated as modulators of renal metabolism of 25-hydroxyvitamin D3 (25(OH)D3). During a 3-h perfusion with 3[H]25(OH)D3, the kidney produced high yields of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) depending on whether the rat had previously been, respectively, normocalcemic, normophosphatemic, vitamin D-replete or hypocalcemic, hypophosphatemic, vitamin D-deplete. With longer perfusion (up to 12 h), kidneys from normocalcemic, normophosphatemic, vitamin D-replete rats mainly produced 24,25(OH)2D3 but also amounts of 1,25(OH)2D3. This pattern was unaltered by reducing Ca or Pi concentrations of perfusate or by adding parathyroid hormone. Kidneys of hypocalcemic, hypophosphatemic, vitamin D-deplete rats perfused with low Ca, low Pi medium for 12 h at first produced 1,25(OH)2D3 exclusively. However, 24,25(OH)2D3 appeared after 4 h and accumulated thereafter, whereas 1,25(OH)2D3 synthesis ceased after 7 h, a metabolic pattern unaffected by the concentration of substrate or end products in the perfusate or by addition of cyclic AMP. The model shows promise for studying regulation of 25(OH)D3 metabolism by the kidney.