Overexpression of Bcl-2 and related anti-apoptotic gene products has been shown to increase the intracellular concentration of the antioxidant tripeptide glutathione in neuronal and hematopoietic cells. A similar examination of HeLa cells that stably overexpress Bcl-2 (Bcl-2/HeLa) demonstrated that the reduced form of glutathione (GSH) was increased by 60% compared to control cells (80 nmol GSH/mg protein compared to 50 nmol GSH/mg). Expression of gamma-glutamylcysteine synthetase, the rate limiting enzyme for glutathione synthesis was found to be independent of Bcl-2 overexpression, as determined by Northern blot analysis and immunoprecipitation of [35-S]-labeled enzyme. Bcl-2 overexpression did not alter the rate of GSH biosynthesis, measured under steady state conditions. Thus, the increase in GSH concentration was not the result of increased synthesis. Two activities have been described which govern efflux of reduced glutathione (GSH), RsGshT known as the sinusoidal transporter and RcGshT, known as the canalicular transporter. Both are low affinity, bidirectional, ATP and Na-independent. Consistent with expression of sinusoidal activity, DTT was found to stimulate GSH efflux while the amino acid methionine inhibited efflux in both HeLa and Bcl-2/HeLa cells. However, methionine-dependent inhibition of efflux was found to be significantly increased by expression of Bcl-2. To test the prediction that the increase in GSH observed in Bcl-2/HeLa cells was mediated by methionine; Bcl-2/HeLa cells were cultured for 24 hrs in methionine-free growth medium. Under these conditions, the GSH concentration of the Bcl-2/HeLa cells dropped to the level observed in HeLa cells (50 nmol GSH/mg protein). These studies suggest that overexpression of Bcl-2 increases GSH levels by altering methionine-dependent GSH efflux, an activity associated in HeLa cells with expression of the RsGshT transporter.