Keratinocytes produce large amounts of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) in vitro. 1,25(OH)2D3 is detectable in anephric humans and pigs and can be increased to near-normal levels by vitamin D or 250HD, indicating an extrarenal source. To determine whether the skin is one of these extrarenal sources, we perfused isolated flaps of porcine skin for 8 h with 250HD3 in serum-free medium at 1 ml/min, collecting the venous effluent as 15-min samples. The samples were extracted and the vitamin D metabolites purified by high-performance liquid chromatography and assayed by competitive protein-binding techniques. Production of 1,25(OH)2D3 continued for the duration of the perfusion, tending to increase in the last 2 hours. The amount of 1,25(OH)2D3 produced varied both with time in the same pig skin and between pig skins; maximum production of 1,25(OH)2D3 in these experiments was 8 pg/min. 24,25(OH)2D3 production was higher than 1,25(OH)2D3 production, reaching a maximum rate of 180 pg/min. Considering that the production rate of 1,25(OH)2D3 in humans is 1.25 ng/min and that a 48-cm2 skin flap represents 1/350 the surface area of a human, and assuming that human and pig skin make 1,25(OH)2D3 at comparable rates, one can determine that the skin has the potential to maintain near-normal levels of 1,25(OH)2D3 in the absence of kidneys when provided with adequate substrate.