Metabolites formed in vivo from [3H]retinoic acid dosed intrajugularly to vitamin A-deficient rats and to vitamin A-deficient rats supplemented orally with unlabeled retinoic acid were investigated. Extracts of liver, small intestinal mucosa, kidney, testes and serum were separated into charged uncharged fractions by DEAE-Sephadex. This allowed the direct application of 20-40% of the combined charged extracts from up to six organs to be loaded onto a high-performance liquid chromatography column. The large aliquot size plus the use of relatively high specific activity [3H]retinoic acid resulted in detection of nanomolar metabolite quantities. The substantial resolution achieved with the high-performance liquid chromatography gradient system aided in demonstrating the complexity, extent and variations of retinoic acid metabolism in vivo, The metabolic profiles changed with retinoic acid pretreatment, time after dose and tissue source. Some 3H-labeled metabolites were predominant in vitamin A-deficient animals; others appeared to be predominant in the retinoic acid-supplemented animals. The gross effect of retinoic acid supplementation was to both accelerate retinoic acid metabolism and cause an accumulation of more polar metabolites. It appears that retinoic acid metabolism in vivo is a complex process that occurs through multiple metabolites, which are, at least partially, tissue-specific.