β-Carotene dioxygenase catalyzes the conversion of provitamin A carotenoids to vitamin A in mammalian tissues. Whether the enzyme can also cleave non-provitamin A carotenoids to retinoid analogs with biological activities is still unclear. We investigated (i) substrate specificities of β-carotene dioxygenase toward provitamin A and non-provitamin A carotenoids and (ii) potential antagonistic effects of non-provitamin A carotenoids on β-carotene conversion to vitamin A. Provitamin A substrates were 8 to 23% as active as β-carotene. No polar metabolites were detected with canthaxanthin or zeaxanthin as substrates; these compounds efficiently inhibited the β-carotene cleavage reaction by 71 and 40%, respectively. Kinetic studies indicated mixed inhibition for canthaxanthin (Ki= 1.6 μM) and non-competitive for zeaxanthin (Ki= 7.8 μM), suggesting that both compounds do not interact significantly with the active site of the enzyme.In vivo,dietary combinations of canthaxanthin and β-carotene resulted in lower liver levels of both carotenoids and vitamin A and in a higher β-carotene/vitamin A ratio as compared to groups supplemented with the compounds separately. This supports the view that canthaxanthin at high doses competes with β-carotene for intestinal absorption and inhibits the conversion of β-carotene to vitamin A. Thus, we suggest that although canthaxanthin is not a substrate for β-carotene dioxygenase, it is likely to affect the activity of provitamin A carotenoids by direct interaction with the enzyme.