The biosynthesis of retinoic acid from beta-apocarotenoic acids was examined for a beta-oxidation-like process using both rabbit liver mitochondrial fractions with various beta-apocarotenoic acids (beta-apo-14'-, beta-apo-12'-, and beta-apo-8'-carotenoic acid) and perfusion in ferret liver through the portal vein with beta-apo-8'-carotenoic acid. The in vitro incubation of beta-apo-8', beta-apo-12'-, and beta-apo-14'-carotenoic acids gave rise to shorter chain beta-apocarotenoic acids as well as retinoic acid. The rate of retinoic acid synthesis from 10 microM beta-apo-8', beta-apo-12'-, and beta-apo-14'-carotenoic acids was 11 +/- 2, 18 +/- 3, and 30 +/- 7 pmol/h/mg of protein, respectively. The stepwise oxidation of beta-apocarotenoic acid in mitochondria was dose-related to both protein concentration and substrate concentration. beta-Apocarotenoic acid oxidation was inhibited in a dose-dependent manner when it was co-incubated with oleoyl-CoA. The in vivo perfusion of ferret liver with beta-apo-8'-carotenoic acid resulted in a linear increase in the retinoic acid concentration of bile, which was completely abolished by co-perfusion of 3-mercaptopropionic acid, an inhibitor of long chain acyl-CoA dehydrogenase, and partially inhibited by 2-tetradecylglycidic acid, an inhibitor of carnitine-palmitoyl-CoA transferase I. However, the formation of retinoic acid from the beta-apocarotenoic acids was not inhibited, either in vitro or in vivo, by citral, an inhibitor of retinal oxidase. Thus, the formation of retinoic acid was not occurring by the central cleavage pathway. These data suggest that the oxidation of intermediate compounds between beta-carotene and retinoic acid may undergo a type of beta-oxidative process to form retinoic acid, which is reminiscent of mitochondrial fatty acid beta-oxidation. This pathway may play an important role in the biosynthesis of retinoic acid from beta-carotene.