Postabsorptive metabolism of [3H]nicotinic acid by rat proximal jejunum was studied in vitro using the everted sac technique and in vivo by applying ligated loops. Metabolites were analyzed by HPLC with radiochemical detection. Within 60 min of incubation nicotinic acid was almost completely metabolized in vivo. Only 3.2% of the label could be detected in the original substrate nicotinic acid, whereas > 90% of the radioactivity present in the gut wall was nicotinamide. Further in vitro experiments revealed that conversion was rapid; after 10 min of incubation > 40% of the substrate was converted to other metabolites. From analysis of the serosal fluid, it was evident that only nicotinic acid and nicotinamide were transferred to the serosal compartment. Rapid conversion of nicotinic acid might affect the transport step itself by metabolic trapping, resulting in the maintenance of a substrate gradient necessary for passive absorption. In contrast, as examined by gradient centrifugation, the substrate in subcellular organelles is transferred only to a minor extent.