Transforming growth factor-beta (TGF-beta) and TGF-beta-related factors induce apoptosis in a variety of tissues; however, the mechanism underlying this induction is largely unknown. Here, we demonstrate that TGF-beta induces the expression of the death-associated protein kinase (DAP-kinase) as an immediate early response in cells that undergo apoptosis in response to TGF-beta. DAP-kinase is a positive mediator of apoptosis induced by certain cytokines and oncogenes. We show that the DAP-kinase promoter is activated by TGF-beta through the action of Smad2, Smad3 and Smad4. Overexpression of DAP-kinase triggers apoptosis in the absence of TGF-beta, whereas inhibition of DAP-kinase activity protects cells from TGF-beta-induced apoptosis, blocks TGF-beta-induced release of cytochrome c from mitochondria and prevents TGF-beta-induced dissipation of the mitochondrial membrane potential. Our findings indicate that DAP-kinase mediates TGF-beta-dependent apoptosis by linking Smads to mitochondrial-based pro-apoptotic events.