Three mechanisms are commonly suggested for the movemerlt of lipids between intracellular organelles: transfer mediated by cytosolic lipid transfer proteins, vesicle-mediated transfer, and transfer via regions of membrane continuity between organelles. The mechanism of translocation of phosphatidylserine from its site of synthesis on the endoplasmic reticulum (and related membranes) to the site of phosphatidylserine decarboxylation in the mitochondria has been investigated. Several experiments indicate that a transfer mediated by soluble cytosolic phospholipid transfer proteins, or by vesicles, is unlikely. Rather, the most likely mode of import of newly-synthesized phosphatidylserine into mitochondria is contact between the endoplasmic reticulum and mitochondrial membranes. In support of this mechanism we have isolated an endoplasmic reticulum-like."mitochondria-associated membrane" fraction and shown that it has some, but not all, properties of the endoplasmic reticulum. The mitochondria-associated membranes are enriched in lipid biosynthetic enzymes, especially phosphatidylserine synthase. When either phosphatidylserine translocation to mitochondria is blocked (by ATP depletion), or phosphatidylserine decarboxylation is blocked (with hydroxylamine), newly-synthesized phosphatidylserine accumulates in the mitochondria-associated membrane but not in microsomes, suggesting that phosphatidylserine traverses the mitochondria-associated membrane on its route from the endoplasmic reticulum to mitochondria.