The metabolism of 25-hydroxyvitamin D3 (25-OHD3) was compared following its intracardial or gastric administration. The rats were deprived of calcium and vitamin D. A mixture of radiolabeled (0.3 microCi) and stable (2 micrograms) 25-OHD3 was given as a single dose. After 24 h the rats given the dose by gastric tube had significantly lower serum concentrations of 25-OHD3 and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] than those injected intracardially. In contrast, serum 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] was much higher in the rats given the 25-OHD3 dose by gastric tube (6.2 nmol/liter +/- 1.3 SD, n = 7) compared to the intracardial group (0.9 nmol/liter +/- 0.5, p less than 0.001). The preceding results were based on specific radioactivity of metabolites. The same findings were obtained by reanalyzing the samples using conventional competitive binding assays for 25-OHD3, 1,25-(OH)2D3, and 24,25-(OH)2D3. The results show that orally administered 25-OHD3 is partly metabolized to 24,25-(OH)2D3 presystemically.