Bax, a member of the Bcl-2 family, directly induces the release of cytochrome c (Cyt c) from isolated mitochondria without requiring caspases. This study demonstrates that recombinant Bax protein, when added to isolated mitochondria, triggers Cyt c release, while a BH3 domain peptide of Bax is ineffective. Neither mitochondria nor Bax alone induce caspase activation in cytosol, but their combination triggers Cyt c release and caspase activation. Supernatants from Bax-treated mitochondria also induce caspase processing and activation. Recombinant Bcl-XL protein blocks Bax-induced Cyt c release and prevents caspase activation. In contrast, broad-specificity caspase inhibitor zVAD-fmk and caspase-inhibiting protein X-IAP do not affect Bax-induced Cyt c release but prevent subsequent caspase activation. Unlike Ca²⁺, which induces mitochondrial swelling and outer membrane rupture, Bax does not cause mitochondrial swelling in vitro, suggesting it uses an alternative mechanism for Cyt c release. Bax-induced Cyt c release occurs independently of mitochondrial permeability transition (PT), indicating that Bax directly triggers Cyt c release without requiring PT. These findings suggest that Bax directly induces Cyt c release from mitochondria, and that Cyt c release precedes caspase-3 processing. The study also shows that Bcl-XL and cyclosporin A (CsA) inhibit Bax-induced Cyt c release, indicating that Bax may interact with components of the mitochondrial megapore. The results highlight that Bax can directly induce Cyt c release without requiring caspases, and that this process does not depend on mitochondrial PT. The findings provide new insights into the mechanisms by which Bax induces apoptosis.Bax, a member of the Bcl-2 family, directly induces the release of cytochrome c (Cyt c) from isolated mitochondria without requiring caspases. This study demonstrates that recombinant Bax protein, when added to isolated mitochondria, triggers Cyt c release, while a BH3 domain peptide of Bax is ineffective. Neither mitochondria nor Bax alone induce caspase activation in cytosol, but their combination triggers Cyt c release and caspase activation. Supernatants from Bax-treated mitochondria also induce caspase processing and activation. Recombinant Bcl-XL protein blocks Bax-induced Cyt c release and prevents caspase activation. In contrast, broad-specificity caspase inhibitor zVAD-fmk and caspase-inhibiting protein X-IAP do not affect Bax-induced Cyt c release but prevent subsequent caspase activation. Unlike Ca²⁺, which induces mitochondrial swelling and outer membrane rupture, Bax does not cause mitochondrial swelling in vitro, suggesting it uses an alternative mechanism for Cyt c release. Bax-induced Cyt c release occurs independently of mitochondrial permeability transition (PT), indicating that Bax directly triggers Cyt c release without requiring PT. These findings suggest that Bax directly induces Cyt c release from mitochondria, and that Cyt c release precedes caspase-3 processing. The study also shows that Bcl-XL and cyclosporin A (CsA) inhibit Bax-induced Cyt c release, indicating that Bax may interact with components of the mitochondrial megapore. The results highlight that Bax can directly induce Cyt c release without requiring caspases, and that this process does not depend on mitochondrial PT. The findings provide new insights into the mechanisms by which Bax induces apoptosis.