The sphingolipid ceramide has been implicated in mediating cell death that is accompanied by mitochondrial functional alterations. Moreover, ceramide has been shown to accumulate in mitochondria upon induction of apoptotic processes. In this study, we sought to evaluate the effects of natural, highly hydrophobic long-chain ceramides on mitochondrial function in vitro. Ceramide in a dodecane/ethanol delivery system inhibited the opening of the mitochondrial permeability transition pore (PTP) induced by either oxidative stress, SH group cross-linking, or high Ca2+ load, suggesting that the inhibitory point is at a level at which major PTP regulatory pathways converge. Moreover, ceramide had no effect on well known mitochondrial components that modulate PTP activity, such as cyclophilin D, voltage-dependent anion channel, adenine nucleotide transporter, and ATP synthase. The inhibitory effect of ceramide on PTP was not stereospecific, nor was there a preference for ceramide over dihydroceramide. However, the effect of ceramide on PTP was significantly influenced by the fatty acid moiety chain length. These studies are the first to show that long-chain ceramide can influence PTP at physiologically relevant concentrations, suggesting that it is the only known potent natural inhibitor of PTP. These results suggest a novel mechanism of ceramide regulation of mitochondrial function.