P lasma 25-hydroxyvitamin D and 24, 25-dihydroxy-vitamin D [24,25-(OH)₂D] concentrations were measured in normal and chronically dialyzed anephric humans and pigs. Measurement of the 24, 25-(OH)₂D was preceded by three purification steps involving one Sephadex LH-20 column and two high-pressure liquid chromatographic columns. The final high-pressure liquid chromatography step involved resolution of 25-hydroxy-vitamin D₃-26,23 lactone and 25,26-dihydroxy-vitamin D₂ from 24,25-dihydroxyvitamin D₂ and 24,25-dihydroxyvitamin D₃ [24,25-(OH)₂D₃]. The total 25-hydroxyvitamin D [25-hydroxyvitamin D₂ plus 25-hydroxyvitamin D₃ (25-OHD₃)] was 31.7±3.6 ng/ml in the plasma of eight anephric human subjects and 40.1±3.7 ng/ml in five normal human subjects. Six of the eight anephric patients had undetectable (<0.2 ng/ml) 24,25-(OH)₂D concentrations. Two of the eight patients had very low (0.51 and 0.41 ng/ml), but detectable, 24,25-dihydroxyvitamin D₂. The normal human volunteers had plasma 24,25-(OH)₂D concentrations of 2.8±0.7 ng/ml. Chronically dialyzed anephric and normal pigs were given intramuscular injections of massive amounts (5 × 10⁶ IU) of vitamin D₃ immediately after surgery (day 0) and again on day 7. In anephric pigs, plasma 25-OHD₃ progressively rose from 12±4 ng/ml on day 0 to 705±62 ng/ml on day 10. The 25-OHD₃ concentrations in normal pigs rose from 8±2 ng/ml on day 0 to 439±64 ng/ml on day 10. Plasma 25-OHD₃ was higher in anephrics throughout the experiment, and concentrations were significantly higher (P < 0.05) on days 9 and 10. Plasma 24,25-(OH)₂D₃ concentrations declined progressively in anephric pigs from 3.6±0.6 ng/ml on day 0 to 3.2±0.7 ng/ml on day 2. During days 4-10, plasma 24,25-(OH)₂D₃ was not apparent until plasma 25-OHD₃ was >400 ng/ml. In control pigs, plasma 24,25-(OH)₂D₃ was elevated from 4.3±0.6 ng/ml on day 0 to 178±2.7 ng/ml on day 3. Plasma 24,25-(OH)₂D₃ was significantly higher (P < 0.05) in controls on days 1-8. At the end of the experiment (day 10), 24,25-(OH)₂D₃ concentrations were similar and not significantly different in both groups (87.0±18.4 ng/ml in anephric and 110.3±32.1 ng/ml in normal pigs). The identity of the 24,25-(OH)₂D₃ isolated from anephric pig plasma was confirmed by mass spectroscopy. Our data suggest that anephric humans receiving normal dietary levels of vitamin D₃ have little or no ability to produce 24,25-(OH)₂D. However, we have shown that pigs produce 24,25-(OH)₂D₃ when plasma 25-OHD₃ is extremely high (>400 ng/ml).