When radiolabeled 1,25-dihydroxyvitamin D3 is added to the perfusion medium of a rat liver perfusion system, a polar metabolite of 1,25-dihydroxyvitamin D3 is formed in the liver and excreted in the bile. Addition of radiolabeled 1,25-dihydroxyvitamin D3 to the perfusion medium without a liver in the circuit results in no formation of the polar metabolite. These data suggest that a polar metabolite of 1,25-dihydroxyvitamin D3 is formed in the liver prior to being excreted in bile. Moreover, it is likely that polar metabolites of 1,25-dihydroxyvitamin D3 present in the bile of rats in vivo are formed in the liver.