Biliary radioactivity excretion was studied in 10 patients with postcholecystectomy T-tube drainage after intravenous administration of 3H-1,25-dihydroxyvitamin D3. The mean +/- SD radioactivity excreted in T-tube bile expressed as a percentage of the administered dose was 18.9 +/- 10.7% per 24 hours. After correction for incomplete bile collection the value obtained was 28.8 +/- 12.8%. The mean chloroform solubility of the biliary radioactivity increased from 17.0 +/- 8.4% to 69.4 +/- 15.1% after incubation with beta-glucuronidase. High performance liquid chromatography of chloroform extracts of bile revealed that most of the eluted radioactivity was more polar than 1,25(OH)2D3. The percentage radioactivity eluting as 3H-1,25(OH)2D3 increased from approximately 2.4 +/- 1.9 to 16.2 +/- 8.0 after incubation with beta-glucuronidase. We conclude that significant amounts of intravenously administered 3H-1,25(OH)2D3 are excreted in bile, mostly as more polar metabolites. The increase in free 3H-1,25(OH)2D3 after incubation with beta-glucuronidase indicates that glucuronides of 1,25(OH)2D3 are present in bile.