CD36, a membrane protein that facilitates fatty acid uptake, is highly expressed in the intestine on the luminal surface of enterocytes. Cd36 null (Cd36 ؊/؊ ) mice exhibit impaired chylomicron secretion but no overall lipid absorption defect. Because chylomicron production is most efficient proximally we examined whether CD36 function is important for proximal lipid absorption. CD36 levels followed a steep decreasing gradient along three equal-length, proximal to distal intestinal segments (S1-S3). Enterocytes isolated from the small intestines of Cd36 ؊/؊ mice, when compared with wild type counterparts, exhibited reduced uptake of fatty acid (50%) and cholesterol (60%) in S1. The high affinity fatty acid uptake component was missing in Cd36 ؊/؊ cells. Fatty acid incorporation into triglyceride and triglyceride secretion were also reduced in Cd36 ؊/؊ S1 enterocytes. In vivo, proximal absorption was monitored using mass spectrometry from oleic acid enrichment of S1 lipids, 90 min (active absorption) and 5 h (steady state) after intragastric olive oil (70% triolein). Oleate enrichment was 50% reduced at 90 min in Cd36 ؊/؊ tissue consistent with defective uptake whereas no differences were measured at 5 h. In Cd36 ؊/؊ S1, mRNA for L-fabp, Dgat1, and apoA-IV was reduced. Protein levels for FATP4, SR-BI, and NPC1L1 were similar, whereas those for apoB48 and apoA-IV were significantly lower. A large increase in NPC1L1 was observed in Cd36 ؊/؊ S2 and S3. The findings support the role of CD36 in proximal absorption of dietary fatty acid and cholesterol for optimal chylomicron formation, whereas CD36-independent mechanisms predominate in distal segments. CD36 or fatty acid translocase (FAT)3 is an 88-kDa transmembrane protein with broad specificity. Its ligands include long-chain fatty acids, native and oxidized lipoproteins, thrombospondin-1, collagen, amyloid ␤, and malaria-infected erythrocytes, recently reviewed in Ref. 1. CD36 has been shown to bind long-chain fatty acids (2, 3) and to facilitate their transfer into the cell (4, 5). Deficiency or overexpression of the protein is associated with alterations in uptake and metabolism of longchain fatty acids in rodents (4, 6, 7). In humans, Cd36 deficiency (8) and polymorphisms in the Cd36 gene (9) are associated with abnormalities in FA clearance (10, 11), insulin responsiveness (11, 12), and lipoprotein metabolism (13, 14). As a result CD36 has been implicated in the etiology of diabetes and atherosclerosis (14, 15).Consistent with its role in FA uptake CD36 is very abundant in the heart, skeletal muscle, adipose tissue (16), and the capillary endothelium (17). The protein is also highly expressed in the small intestine (16,18,19) and localizes to the apical membrane of villi enterocytes (19). Expression levels and localization strongly suggest a function in lipid absorption but this could not be documented in previous studies by us and others (20 -22). Administration of a lipid load to Cd36-deficient mice did not identify alterations in the blood appearance of intes...