Rat intestinal mucosa was prepared and incubated with beta-carotene by the procedure of Goodman and Olson [Goodman, DeW. S., & Olson, J.A. (1969) Methods Enzymol. 15, 462-475] to determine beta-carotene cleavage activity. A new detection system for the reaction products of the described enzyme beta-carotene 15,15'-dioxygenase (EC 1.13.11.21) employs solvent extraction of retinoids and carotenoids followed by high-performance liquid chromatography separation and photometric detection of the pigments. It has not detected any newly formed retinal or other retinoids in the intestinal protein preparations from normal or vitamin A deficient rats. The latter were chosen as a possible source of more active enzyme preparations. With corresponding blank samples subjected to identical conditions of incubation but without added protein, small amounts of beta-apocarotenals could be detected. They were previously reported as cleavage products of beta-carotene [Ganguly, J., & Sastry, P.S. (1985) World Rev. Nutr. Diet. 45, 198-220] but are clearly not formed as a result of an enzymatic reaction. The failure to detect in vitro enzymatic central or random cleavage of the beta-carotene molecule in extracts of rat intestinal mucosa emphasizes the need to reevaluate the existing theory of conversion of beta-carotene into vitamin A.