Pretreatment by L-buthionine sulfoximine (BSO), which inactivates gamma-glutamylcysteine synthetase and, therefore, inhibits glutathione (GSH) synthesis, greatly increased the sensitivity of tumor cells to the antiproliferative effects of several NO-donating compounds. The sensitization that resulted from depletion of cellular GSH pools was observed in tumor cells exhibiting different degrees of resistance to NO. In contrast, GSH depletion of tumor target cells did not affect their sensitivity to the cytostatic activity of activated macrophages and other NO-producing cells (EMT6 cells treated by interferon gamma and LPS). The kinetics for NO generation is a parameter that may differentiate NO-producing cells and short-lived NO donors. To study the relationship between the magnitude of NO fluxes and the increased toxicity on BSO-pretreated cells, two NO-releasing zwitterions derived from polyamines (NONOates) with different half-lives were selected. NO fluxes as a function of time were simulated, according to the donor half-life and initial concentration, and antiproliferative effects on control and BSO-treated cells were compared. GSH depletion increased the sensitivity of tumor cells in the case of the less stable NO donor only. We, thus, propose that intracellular GSH is specifically protective against high fluxes of NO.