Glutamate cysteine ligase (GCL), which synthesizes gamma-glutamyl-cysteine (gamma-GC), is the rate-limiting enzyme in GSH biosynthesis. gamma-GC may be produced by the catalytic subunit GCLC or by the holoenzyme (GCLholo), which comprises GCLC and the modifier subunit GCLM. The Gclm(-/-) knock-out mouse shows tissue levels of GSH that are between 9 and 40% of the Gclm(+/+) wild-type mouse. In the present study, we used recombinant GCLC and GCLM and Gclm(-/-) mice to examine the role of GCLM on gamma-GC synthesis by GCLholo. GCLM decreased the Km for ATP by approximately 6-fold and, similar to other species, decreased the Km for glutamate and increased the Ki for feedback inhibition by GSH. Furthermore, GCLM increased by 4.4-fold the Kcat for gamma-GC synthesis; this difference in catalytic efficiency of GCLholo versus GCLC allowed us to derive a mathematical relationship for gamma-GC production and to determine the relative levels of GCLholo and GCLC; in homogenates of brain, liver, and lung, the ratio of GCLC to GCLholo was 7.0, 2.0, and 3.5, respectively. In kidney, however, the relationship between GCLC and GCLholo was complicated. Kidney contains GCLholo, free GCLC, and free GCLM, and free GCLC in kidney cannot interact with GCLM. Taken together, we conclude that, in most tissues, GCLM is limiting, suggesting that an increase in GCLM alone would increase gamma-GC synthesis. On the other hand, our results from kidney suggest that gamma-GC synthesis may be controlled post-translationally.