Ethanol intake depletes the mitochondrial pool of reduced glutathione (GSH) by impairing the transport of GSH from cytosol into mitochondria. S-Adenosyl-L-methionine (SAM) supplementation of ethanol-fed rats restores the mitochondrial pool of GSH. The purpose of the current study was to determine the effect of ethanol feeding on the kinetic parameters of mitochondrial GSH transport, the fluidity of mitochondria, and the effect of SAM on these changes. Male Sprague-Dawley rats were fed ethanol-liquid diet for 4 weeks supplemented with either SAM or N-acetylcysteine (NAC). SAM-supplementation of ethanol-fed rats restored the mitochondrial GSH pool but NAC administration did not. Kinetic studies of GSH transport in isolated mitochondria revealed two saturable, adenosine triphosphate (ATP)-stimulated components that were affected significantly by chronic ethanol feeding: lowering Vmax (0.22 and 1.6 in ethanol case vs. 0.44 and 2.7 nmol/15 sec/mg protein in controls) for both low and high affinity components with the latter showing an increased Km (15.5 vs. 8.9, mmol/L in ethanol vs. control). Mitochondria from SAM-supplemented ethanol-fed rats showed kinetic features of GSH transport similar to control mitochondria. Determination of membrane fluidity revealed an increased order parameter in ethanol compared with control mitochondria, which was restricted to the polar head groups of the bilayer and was prevented by SAM but not NAC supplementation of ethanol-fed rats. The changes elicited in mitochondria by ethanol were confined to the inner membrane; mitoplasts from ethanol-fed rats showed features similar to those of intact mitochondria such as impaired transport of GSH and increased order parameter. A different mitochondrial transporter, adenosine diphosphate (ADP)/ATP translocator, was unaffected by ethanol feeding. Furthermore, fluidization of mitochondria or mitoplasts from ethanol-fed rats by treatment with a fatty acid derivative restored their ability to transport GSH to control levels. Thus, ethanol-induced impaired transport of GSH into mitochondria is selective, mediated by decreased fluidity of the mitochondrial inner membrane, and prevented by SAM treatment.