Most members of the Bcl-2 protein family of apoptosis regulating proteins contain two evolutionarily conserved domains, termed BH1 and BH2. Both BH1 and BH2 in the Bcl-2 protein are required for its function as an inhibitor of cell death and for heterodimerization with the proapoptotic protein Bax. In this report, we mapped the region in Bax required for heterodimerization with Bcl-2 and homodimerization with Bax, using yeast two-hybrid and in vitro protein-protein interaction assays. Neither the BH1 nor the BH2 domain of Bax was required for binding to the wild-type Bcl-2 and Bax proteins. Moreover, Bax (deltaBH1) and Bax (deltaBH2) mutant proteins bound efficiently to themselves and each other, further confirming the lack of requirement for BH1 and BH2 for Bax/Bax homodimerization. Bax/Bax homodimerization was not dependent on the inclusion of the NH2-terminal 58 amino acids of the Bax protein in each dimerization partner, unlike Bcl-2/Bcl-2 homodimers which involve head-to-tail interactions between the region of Bcl-2 where BH1 and BH2 resides, and an NH2-terminal domain in Bcl-2 that contains another domain BH4 which is conserved among antiapoptotic members of the Bcl-2 family. Similarly, heterodimerization with Bcl-2 occurred without the NH2-terminal domain of either Bax or Bcl-2, suggesting a tail-to-tail interaction. The essential region in Bax required for both homodimerization with Bax and heterodimerization with Bcl-2 was mapped to residues 59-101. This region in Bax contains a stretch of 15 amino acids that is highly homologous in several members of the Bcl-2 protein family, suggesting the existence of a novel functional domain which we have termed BH3. Deletion of this 15-amino acid region abolished the ability of Bax to dimerize with itself and to heterodimerize with Bcl-2. The findings suggest that the structural features of Bax and Bcl-2 that allow them to participate in homo-and heterodimerization phenomena are markedly different, despite their amino-acid sequence similarity.