Extracts of rat and rabbit intestinal mucosa were fractionated with ammonium sulfate. The precipitate contained beta-apocarotenoid-14',13'-dioxygenase (ADO) activity. beta-Apo-14'-carotenal was found to be the product of enzymatic cleavage of beta-apo-8'-carotenol. Activities of ADO and beta-carotene-15,15'-dioxygenase (CDO) were detected in the presence of thiols and were inactivated by 1,10-phenanthroline. Optimal pH values were 7.0 for ADO and 8.0 for CDO. Heating at 52 degrees C inhibited ADO by 70% and produced no effect on CDO. ADO activity was maximal in the presence of sodium cholate or 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS). Sodium dodecylsulfate was required for maximal CDO activity. Proteins with ADO activity were not retained by phenyl-Sepharose CL-4B. CDO activity was eluted from columns only in the presence of detergents. The data suggest that the enzymes that catalyze the oxidative cleavage of beta-carotene to yield retinal are different from those that cleave beta-apo-8'-carotenol to yield beta-apo-14'-carotenal.