Glutathione (GSH) is involved in many cellular functions, including cell growth and differentiation. GSH also plays an important role in the protection of cells against oxidative damage and hence in determining the sensitivity of cells to the cytotoxicity of anticancer agents. Because of this, induction of GSH depletion has been proposed as a good strategy for sensitizing tumor cells to antitumor agents. The aim of the present work is to study the effect of buthionine sulfoximine (BSO, a specific cellular GSH-depleting agent) in two rat tumor cell lines derived from the same rhabdomyosarcoma tumor model, the moderately differentiated and low metastatic F21 cell line, and the poorly differentiated and high metastatic S4MH cell line, to investigate the influence of the degree of differentiation in the induction of GSH depletion-based therapy. We observed that, whereas in the S4MH cell line BSO induced a dose-dependent inhibition of both cell growthin vitroand tumorigenic potentialin vivo, in F21 cells the administration of moderate doses of BSO enhanced tumor growth and only at high doses was there a slight reduction of their tumorigenic potential. These effects were in consonance with the fact that the activity of&ggr;-glutamyltranspeptidase (&ggr;-GT) present in the F21 cells was 4 times higher than in the S4MH cells. Indeed, inhibition of&ggr;-GT activity by acivicin not only abrogated the BSO-induced increase of GSH content and of cell growth, but also the combination of acivicin + BSO significantly decreased intracellular GSH levels and cell proliferation, and induced F21 cells to apoptosis. These studies suggest that, as occurs in the rhabdomyosarcoma tumor model,&ggr;-GT levels and the degree of differentiation of tumor cells might influence the response of tumor cells to inducers of GSH depletion, and should be taken into account in therapies based on GSH metabolism.