A 13 kDa protein was isolated from rabbit small intestine brush-border membrane vesicles that was postulated to be involved in intestinal phosphatidylcholine (PC) and cholesterol uptake. This protein has cholesterol and PC-transfer activity in vitro (Turnhofer, H. et al. (1991) Biochim. Biophys. Acta 1064, 275-286) and has a molecular mass and isoelectric point similar to that of the non-specific lipid transfer protein (nsL-TP, identical to sterol carrier protein-2). In addition, the first 28 N-terminal amino acid residues of the 13 kDa protein are nearly identical to nsL-TP from different species (Lipka, G. et al. (1995) J. Biol. Chem. 270, 5917-5925). In view of its possible role in intestinal lipid absorption, the localization of nsL-TP in rat small intestine was investigated using immunohistochemistry and immunoblotting. It is shown that nsLTP is predominantly localized in a subapical zone of the enterocyte but not in the brush-border membrane, thereby excluding a role in lipid uptake of this protein at the level of the plasma membrane. nsL-TP co-localized with the peroxisomal marker PMP70, underscoring earlier observations that nsL-TP is a peroxisomal protein. nsL-TP was found to be present along the entire length of the small intestine. The 58 kDa cross-reactive protein that was recently identified as a peroxisomal thiolase was shown to be present only in a small segment approximately halfway down the jejunum. The close apposition of the peroxisomes with the apical membrane and the discrete distribution of the 58 kDa protein may indicate that these organelles play a role in the intracellular processing of absorbed lipids.