Increased mitochondrial ceramide levels are associated with the initiation of apoptosis. There is evidence that ceramide is causal. Thus, the conversion of the precursor, dihydroceramide, to ceramide by the enzyme dihydroceramide desaturase may be important in preparing the cell for apoptosis. Ceramide can initiate apoptosis by permeabilizing the mitochondrial outer membrane to apoptosis-inducing proteins. However, the mitochondrion's ability to produce ceramide may be limited by its proteome. Here, we show that ceramide synthesized in isolated mammalian endoplasmic reticulum (ER) vesicles from either C8-dihydroceramide or sphingosine to produce long-chain ceramide can transfer to isolated mitochondria. The rate of transfer is consistent with a simple collision model. The transfer of the long-chain ceramide is faster than expected for an uncatalyzed process. Sufficient ceramide is transferred to permeabilize the outer membrane to cytochrome c and adenylate kinase. The mitochondria-associated membranes, ER-like membranes that are tightly associated with isolated mitochondria, can produce enough ceramide to permeabilize the outer membrane transiently. Thus, this ceramide exchange obviates the need for a complete ceramide de novo pathway in mitochondria to increase ceramide levels to the critical value required for functional changes, such as ceramide channel self-assembly followed by protein release.