In mammalian cells, non receptor-mediated apoptosis occurs via the cytochrome c–dependent assembly of a ∼700-kd apoptotic protease-activating factor 1 (Apaf-1)/caspase-9 containing apoptosome complex. This initiates the postmitochondrial-mediated effector caspase cascade. We now show that receptor mediated transforming growth factor β1(TGF-β1)–induced apoptosis in rat hepatoma cells is accompanied by processing and activation of caspases-2, -3, -7, and -8. Furthermore, we show that caspase activation is mediated via the release of cytochrome c and the oligomerization of Apaf-1 into an ∼700-kd apoptosome complex. Similarly, in vitroactivation of hepatoma cell lysates with 2′-deoxyadenosine 5′-triphosphate (dATP) results in the formation of the ∼700-kd apoptosome complex, which recruits and processes caspases-3 and -7. Z-VAD.FMK [benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone], the pan-caspase inhibitor totally inhibits dATP-stimulated caspase activation but does not block the assembly of the large Apaf-1 containing apoptosome complex. However, the recruitment and subsequent processing of caspases-3 and -7 to the apoptosome is blocked. Similarly, in intact cells, although Z-VAD.FMK blocked TGF-β1–induced apoptosis, it did not prevent the oligomerization of Apaf-1 into the apoptosome. However, recruitment and processing of caspases-3 and -7 were prevented by Z-VAD.FMK. These data show that TGF-β1 induces apoptosis via release of cytochrome c and activation of the Apaf-1 apoptosome complex, which initiates the caspase cascade.