The glutathione (GSH) level of CC531 rat colorectal cancer cells is readily decreased by exposure to buthionine sulfoximine (BSO), an inhibitor of GSH synthesis; at 25 microM BSO, these cells died in a non-apoptotic fashion. By continuous exposure of CC531 cells to increasing concentrations of BSO, we obtained a BSO-resistant cell line (CCBR25) that was 50 times more resistant to BSO than the parental cell line. Whereas the GSH content of CCBR25 and CC531 cells was similar, the former contained a much higher level of the Bcl-2 protein. After stable transfection of CC531 cells with the human bcl-2 gene, the resulting Bcl-2-overexpressing cell line appeared to be 9 times more resistant to BSO than the parental cell line. These findings suggest that the Bcl-2 protein offers resistance against the cytotoxic effect of severe GSH depletion.