βCarotene-15,15′-dioxygenase (EC 126.96.36.199; β-carotene dioxygenase) activity in extracts from guinea-pig intestinal mucosa was assayed by supplying [15,15′-14C2l- or [15,15′-3H2]β-carotene dissolved in Tween 80. Methods were developed to minimize the breakdown of labelled β-carotene and β-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 β-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 up-carotenals isolated in these experiments and the formation of between 1.5 and 2 mol retinaldehyde/mol β-carotene consumed confirm the central cleavage mechanism for the enzyme's action. More β-carotene dioxygenase activity was obtained from guinea-pig mucosa than from chicken or pig intestinal mucosa. The β-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 formedlmg protein per h. The formation of a lipid-protein aggregate containing the β-carotene dioxygenase activity, which has been reported to be present in the exclusion volume of Sephadex columns, was avoided if the mucosal serapings were homogenized in buffer at a proportion of 1:4 (w/v).