beta-Carotene-15,15'-dioxygenase (EC 1.13.11.21; beta-carotene dioxygenase) activity in extracts from guinea-pig intestinal mucosa was assayed by supplying [15,15'-14C2]- or [15,15'-3H2] beta-carotene dissolved in Tween 80. Methods were developed to minimize the breakdown of labelled beta-carotene and beta-carotene cleavage products during the isolation procedure. Antioxidants and unlabelled carriers were added to extracting solvents and C18 Sep-Pak cartridges were used to isolate the remaining beta-carotene and retinaldehyde, which was the only cleavage product detected. The labelled material produced by the enzyme was analysed by either normal-phase TLC or reversed-phase HPLC and characterized chemically as retinaldehyde. The lack of other labelled apo-carotenals isolated in these experiments and the formation of between 1.5 and 2 mol retinaldehyde/mol beta-carotene consumed confirm the central cleavage mechanism for the enzyme's action. More beta-carotene dioxygenase activity was obtained from guinea-pig mucosa than from chicken or pig intestinal mucosa. The beta-carotene dioxygenase was obtained as a soluble enzyme which was partially purified by gel filtration and ion-exchange chromatography to a specific activity of 0.6 nmol retinaldehyde formed/mg protein per h. The formation of a lipid-protein aggregate containing the beta-carotene dioxygenase activity, which has been reported to be present in the exclusion volume of Sephadex columns, was avoided if the mucosal scrapings were homogenized in buffer at a proportion of 1:4 (w/v).