Endoplasmic reticulum-like membranes (MAM) that are associated with mitochondria have been implicated as intermediates in the import of lipids, particularly phosphatidylserine, from the endoplasmic reticulum to mitochondria (Vance, J.E. (1990) J. Biol. Chem. 265, 7248-7256; Shiao, Y.-J. et al. (1995) J. Biol. Chem. 270, 11190-11198). We have now examined further the role of MAM in lipid metabolism using the mnd/mnd mouse, a model for the human degenerative disease neuronal ceroid lipofuscinosis. The biochemical phenotype of the mnd/mnd mutant mouse (in which lipids and proteins accumulate abnormally in storage bodies in cells of affected tissues) suggested that the mutation might lead to impaired mitochondrial import of lipids and proteins as a result of a defective linkage between MAM and mitochondria. We, therefore, investigated the status of MAM and phospholipid metabolism in mnd/mnd mice livers. Separation of MAM from livers of older, but not younger, mnd/mnd mice was aberrant. In addition, the amount of the MAM-specific protein, phosphatidylethanolamine N-methyltransferase-2 (PEMT2), was greatly reduced in homogenates and MAM from livers of mnd/mnd mice of all ages, although PEMT2 mRNA abundance was normal. Moreover, PEMT activity in MAM from mnd/mnd mice was 60% less than in control mice. Activities of two additional phospholipid biosynthetic enzymes-CTP:phosphocholine cytidylyltransferase and phosphatidylserine synthase-were also reduced by > 50% in mnd/mnd microsomes. Radiolabeling experiments in hepatocytes indicated that neither the mitochondrial import nor the subsequent metabolism of phosphatidylserine was grossly affected in mnd/mnd mice. However, 3 proteins (cytochrome b5, NADH:cytochrome b5 reductase and mitochondrial F1Fzero-ATP synthase c subunit) which are normally present in mitochondria were partially redistributed to microsomes in mnd/mnd mouse liver. These studies indicate that MAM are defective in the mnd/mnd mutant mouse in which the biochemical phenotype includes an abnormal accumulation of lipids and proteins in storage bodies.