Bax, a member of the Bcl-2 family of proteins, has been shown to promote apoptosis while other members of the family, including Bcl-XL and Bcl-2, inhibit cell death induced by a variety of stimuli. The mechanism by which Bax promotes cell death is poorly understood. In the present report, we assessed the ability of Bax to antagonize the death repressor activity of Bcl-XL during chemotherapy-induced apoptosis in the lymphoid cell line, FL5.12. Expression of wild-type Bax countered the repressor activity of Bcl-XL against cell death mediated by VP-16 and cisplatin. We performed site-directed mutagenesis of the BH1, BH2, and BH3 homology regions in Bax to determine the ability of wild-type and mutant Bax to heterodimerize with Bcl-XL and to antagonize the protective effect of Bcl-XL against chemotherapy-induced apoptosis. Bax proteins expressing alanine substitutions of the highly conserved amino acids glycine 108 in BH1, tryptophan 151 and 158 in BH2, and glycine 67 and aspartic acid 68 in BH3 retained their ability to promote chemotherapy-induced cell death that was inhibited by Bcl-XL and to form heterodimers with Bcl-XL. Bax proteins containing deletions of the most highly conserved amino acids in BH1 (Delta102-112) and BH2 (Delta151-159) maintained the ability of Bax to antagonize the death repressor activity of Bcl-XL and to associate with Bcl-XL. However, Bax with BH3 deleted did not form heterodimers with Bcl-XL, but retained its ability to counter the death repressor activity of Bcl-XL. These results demonstrate that the conserved BH3, but not BH1 or BH2, homology region of Bax is necessary for its interaction with Bcl-XL in mammalian cells. Furthermore, our results indicate that Bax does not require BH1, BH2, BH3, or heterodimerization with Bcl-XL to counter the death repressor activity of Bcl-XL. Therefore, Bax can antagonize Bcl-XL during VP-16 and, in a lesser degree, during cisplatin-induced cell death independent of its heterodimerization with Bcl-XL.