The purposes of this study were to determine the location of beta-carotene dioxygenase (EC 1.13.11.21) activity within the rat gastrointestinal tract, within the villus and within enterocytes, and to identify the metabolites produced in each intestinal fraction. In Wistar female rats, maximal activity was detected in the cytosol (74-93% of the total cellular activity) of mature functional enterocytes harvested from the jejunum (67% of the intestinal activity). The specific activity, expressed in pmol of retinoids/(h x mg protein) rose from 49 +/- 3 in the stem cells to 199 +/- 12 in the mature functional cells (P < 0.05). Thus the intestinal beta-carotene cleavage activity might be regulated during the enterocyte maturation process. By using HPLC with diode array and radioactive detectors, retinal, and in the presence of NAD+, retinoic acid, were identified as the only metabolites produced. No beta-12'-, 10'-, and 8'-apo-carotenals were detected, even when various enzyme sources were tested. These results suggest that the major, if not the sole, pathway for the formation of vitamin A from beta-carotene in the rat intestine is central cleavage.