Membrane association between mitochondria and the endoplasmic reticulum of the yeast Saccharomyces cerevisiae is probably a prerequisite for phospholipid translocation between these two organelles. This association was visualized by fluorescence microscopy and computer-aided three-dimensional reconstruction of electron micrographs from serial ultrathin sections of yeast cells. A mitochondria-associated membrane (MAM), which is a subfraction of the endoplasmic reticulum, was isolated and re-associated with mitochondria in vitro. In the reconstituted system, phosphatidylserine synthesized in MAM was imported into mitochondria independently of cytosolic factors, bivalent cations, ATP, and ongoing synthesis of phosphatidylserine. Proteolysis of mitochondrial surface proteins by treatment with proteinase K reduced the capacity to import phosphatidylserine. Phosphatidylethanolamine formed in mitochondria by decarboxylation of phosphatidylserine is exported to the endoplasmic reticulum where part of it is converted into phosphatidylcholine. In contrast with previous observations with permeabilized yeast cells [Achleitner, G., Zweytick, D., Trotter, P., Voelker, D. & Daum, G. (1995) J. Biol. Chem. 270, 29836-29842], export of phosphatidylethanolamine from mitochondria to the endoplasmic reticulum was shown to be energy-independent in the reconstituted yeast system.