An endoplasmic reticulum-like membrane fraction, termed the “mitochondria-associated membrane” (MAM), that co-isolates with mitochondria from rat liver has been characterized. One potential function of the MAM is as a membrane bridge between the endoplasmic reticulum and mitochondria that may be involved in transfer of phospholipids between these two organelles (Vance, J. E. (1990) J. Biol. Chem. 265, 7248-7256). A polyclonal antibody directed against a peptide corresponding to the C terminus of phosphatidylethanolamine N-methyltransferase-2, a specific marker protein of the MAM (Cui, Z., Vance, J. E., Chen, M. H., Voelker, D. R., and Vance, D. E. (1993) J. Biol. Chem. 268, 16655-16663), was used in immunofluorescence and immunogold electron microscopy localization studies in rat hepatocytes. Immunoreactive protein was clustered in regions of the cell that did not correspond to the bulk of endoplasmic reticulum. A second potential role for the MAM, as a component of the secretory pathway that supplies lipids for assembly into very low density lipoproteins, has been examined. The MAM contains enzymes of similar, or higher, specific activities to those enzymes in the endoplasmic reticulum for the synthesis of phospholipids, triacylglycerols, cholesterol, and cholesteryl esters. Specific activities of diacylglycerol acyltransferase, acyl-coenzyme A:cholesterol acyltransferase, and phosphatidylserine synthase (base exchange enzyme) are enriched 2.2-3.4-fold in the MAM compared with endoplasmic reticulum. In addition, the microsomal triacylglycerol transfer protein, which is required for the assembly/secretion of apolipoprotein B-containing lipoproteins, was present in the MAM. Nascent apolipoprotein B-containing lipoproteins were isolated from the lumen of the MAM. These lipoproteins had the same average density and composition as nascent apolipoprotein B-containing lipoproteins isolated from heavy and light endoplasmic reticulum fractions, from the Golgi, and lipoproteins newly secreted by cultured rat hepatocytes. The MAM is a pre-Golgi compartment of the secretory route, as shown by pulse-chase studies with apolipoprotein B and albumin, as well as the sensitivity of luminal apolipoprotein B to endoglycosidase H.