Glutathione (GSH) is involved in diverse activities essential for cellular health. Mice lacking the glutamate-cysteine ligase modifier subunit gene (Gclm(–/–)) are severely deficient in GSH and have diminished fecundity, yet are viable. Since depletion of mitochondrial GSH (mt-GSH) is associated with cell death, hepatic mt-GSH homeostasis in C57BL/6J Gclm(–/–) mice and littermate wild-type (WT) controls was evaluated. The cytosolic levels of GSH in Gclm(–/–) mice were only 15% of those found in WT mice, while the mitochondrial GSH levels in Gclm(–/–) mice were 67% of WT levels. In isolated mitochondria, mt-GSH was stable for 12 h at 0 °C, but released by chemical thiols in a membrane potential and temperature-insensitive manner. The order of effectiveness of mt-GSH release by chemicals was 2-mercaptoethanol > dithiothreitol > GSH-ethyl ester > dimercaptosuccininic acid. These results suggest that mt-GSH is bound within the matrix, rather than freely soluble. mt-GSH uptake kinetics helped to explain the relatively higher levelsof mt-GSH in Gclm(–/–) mice (V_m = 6.4 nmol/min mg; K_m = 0.9 mM) versus WT mice (V_m = 5.2 nmol/min mg; K_m = 3.6 mM). GSH transport was competitively inhibited by L-glutamate, with K_i = 1.5 mM for Gclm(–/–) mice versus 3.3 mM for WT mice. The transport proteins responsible for mitochondrial GSH uptake were further characterized employing inhibitors of the known inner mitochondrial membrane GSH transporters, Slc25a10 (dicarboxylate) and Slc25a11 (oxoglutarate). Slc25a10/11 inhibitors averaged about 35% and 20% inhibition in mitochondria from WT and Gclm(–/–) mice, respectively. In contrast, L-glutamate inhibited GSH transport by 7% and 45% in mitochondria from WT and Gclm(–/–) mice, respectively. There were no transcriptional changes in any mitochondrial carrier family gene (Slc25a). It is concluded that, in Gclm(–/–) mice, mt-GSH is preserved due to binding in the mitochondrial matrix and elevated GSH uptake, possibly due in part to the presence of a novel GSH transporter.