Free fatty acids (FFA) and sn-2-monoacylglycerol (MG), the two major hydrolysis products of dietary triacylglycerol (TG), are absorbed from the lumen into polarized enterocytes that line the small intestine. Intensive studies regarding FFA metabolism in the intestine have been published; however, little is known regarding the metabolism of MG. In these studies, we examined the metabolism of sn-2-monoolein (sn-2-18:1) by human intestinal Caco-2 cells. To mimic the physiological presentation of MG to the enterocyte, the metabolism of [ 3 H]sn-2-monoolein was examined by adding taurocholatemixed sn-2-18:1 and albumin-bound sn-2-18:1 at the apical (AP) and basolateral (BL) surfaces of the Caco-2 cell, respectively. The results demonstrate that more sn-2-18:1 was incorporated into TG from AP taurocholate-mixed sn-2-18:1, whereas more phospholipid was synthesized from BL albumin-bound sn-2-18:1. The TG: phospholipid ratio was ϳ5-fold higher for AP relative to BL MG incubation. Qualitatively similar results were observed for bovine serum albumin-bound MG added at the apical surface. It was also found that substantial sn-2-18:1 radioactivity was recovered in the FFA fraction, suggesting that sn-2-18:1 may be directly hydrolyzed within the Caco-2. We therefore used reverse transcription-PCR with primers designed from the murine MG lipase (MGL) gene, and detected the presence of MG lipase mRNA in Caco-2. The human MGL gene was cloned and found to be 83% identical to the murine MGL, and identical to a previously described lysophospholipase-like protein. Northern blot analysis showed the expression of MGL throughout Caco-2 differentiation. Thus, MG metabolism in Caco-2 cells may include not only well established anabolic processes, but also catabolic processes. Further, the observed polarity of MG metabolism suggests that, as for fatty acids, separate precursor and/or product pools of lipid may exist in the intestinal enterocyte.sn-2-Monoacylglycerol (sn-2-MG) 1 and fatty acids (FFA) are the products of pancreatic lipase hydrolysis of dietary triacylglycerol (TG). They are absorbed from the lumen into polarized enterocytes that line the small intestine (1). Following absorption, across the apical (AP) surface of the enterocyte, sn-2-MG and FFA are reincorporated into TG, which are subsequently secreted as the major component of chylomicrons (CM) into the lymphatic system (1). FFA are also taken up across the basolateral (BL) surface of the enterocyte (2), and we have shown recently that MG is taken up across the BL surface of Caco-2 cells as well (3). CM and other TG-rich lipoproteins are hydrolyzed by lipoprotein lipase (LPL) that extends into the vascular space from the capillary endothelial cells of extrahepatic tissues. LPL catalyzes the release of FFA from TG, and circulating TG-rich lipoproteins have been reported to accumulate MG after LPL hydrolysis (4, 5). Further, serum albumin was found to bind sn-2-monoolein (sn-2-18:1) with an apparent dissociation constant (K d ) of ϳ0.2 M (6), and it was shown that albumin-bo...