Buthionine sulfoximine (BSO) selectively inhibits glutathione (GSH) synthesis and has been used to sensitize tumor cells to alkylating agents, but has minimal single-agent cytotoxicity for most cell types. We determined the cytotoxicity of BSO for 18 (12 MYCN amplified; 6 MYCN nonamplified) human neuroblastoma cell lines using DIMSCAN, a digital image microscopy cytotoxicity assay. D-L(R:S) BSO was highly cytotoxic (>3 logs of cell kill) for most neuroblastoma cell lines, with 17/18 cell lines having IC90 values (range 2.1> 1000 μM) below equivalent steady state plasma levels of L(R:S) BSO reported in adult human trials. Cell lines with genomic amplification of MYCN were more sensitive to BSO than MYCN nonamplified cell lines (P = 0.04). D-L(R:S) BSO (500 μM for 72 h) induced apoptosis as detected by DNA laddering, nuclear morphology, and TUNEL staining of DNA fragments using flow cytometry. Maximal cell killing occurred within 48 h and was antagonized by the addition of antioxidants (GSH, vitamin E, and ascorbate). Interestingly, ascorbate had a bimodal effect, with lower doses reversing and higher doses enhancing BSO cytotoxicity. Depletion of GSH in neuroblastoma cells by BSO resulted in increased formation of reactive oxygen species (ROS), as measured by dichlorofluorescein diacetate and flow cytometry. Thus, neuroblastoma cell lines rely on GSH as an antioxidant to counter endogenous production of ROS, and BSO-mediated GSH depletion may be of therapeutic value in neuroblastoma.