The metabolism of vitamin D3 was studied in bilaterally nephrectomized pigs and cotnrol pigs maintained for 8-10 days by daily peritoneal dialysis. Two or three pharmacological doses of vitamin D3 were given im, one on day 0 and the others on days, 4, 6, or 7 of the 8- to 10-day experimental periods. Nephrectomized pigs had extremely high plasma concentrations of 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] and 25,26-(OH)2D3 at the end of the 8- to 10-day periods; similar levels were present in control pigs. However, nephrectomized pigs had no detectable increase in plasma 25-hydroxyvitamin D3 (25OHD3)-26,23-lactone concentrations throughout the experiment, in contrast with the high plasma concentrations of 25OHD3-26,23-lactone in the control pigs. Much higher plasma 25OHD3 concentrations were present in the nephrectomized pigs than in the control pigs. Plasma 1,25-(OH)2D concentrations in nephrectomized and control pigs were similar during most of the experiment, except the anephric pigs usually had significantly higher plasma 1,25-(OH)2D concentrations for several days after the vitamin D3 injections. These studies demonstrate extrarenal production of 24,25-(OH)2D3, 25,26-(OH)2D3, and possibly 1,25-(OH)2D, and kidney-dependent synthesis of 25OHD3-26,23-lactone in the pigs.