Freshly isolated tightly coupled rabbit renal cortical mitochondria rapidly accumulated glutathione (GSH) against an electrical and concentration gradient, and in the presence and absence of pyruvate/malate, succinate, antimycin A, or FCCP. Mitochondrial GSH uptake was dependent on medium GSH concentration, was not saturable, and reached equilibrium within 1 min of addition. Mitochondrial GSH uptake was partially inhibited by glycine, ophthalmic acid, and serine but not glutamate, cysteine, gamma-glutamyl-glutamate, or proline. These results show that 1) mitochondrial GSH uptake is by both a carrier-mediated process and by diffusion, and 2) the GSH carrier system has structural specificity with the glycine residue being a recognition site.