To identify protein components of the intestinal cholesterol transporter, rabbit small intestinal brush border membrane vesicles were submitted to photoaffinity labeling using photoreactive derivatives of 2-azetidinone cholesterol absorption inhibitors. An integral membrane protein of M(r) 145.3+/-7.5 kDa was specifically labeled in brush border membrane vesicles from rabbit jejunum and ileum. Its labeling was concentration-dependently inhibited by the presence of cholesterol absorption inhibitors whereas bile acids, D-glucose, fatty acids or cephalexin had no effect. The inhibitory potency of 2-azetidinones to inhibit photolabeling of the 145 kDa protein correlated with their in vivo activity to inhibit intestinal cholesterol absorption. These results suggest that an integral membrane protein of M(r) 145 kDa is (a component of) the cholesterol absorption system in the brush border membrane of small intestinal enterocytes.