Tumor necrosis factor (TNF)-alpha has been shown to induce apoptosis in a variety of cell types including cardiac myocytes. Sphingosine/ceramide and nitric oxide have been associated with apoptosis induced by TNF-alpha; however, signaling mechanisms of TNF-alpha-induced apoptosis in cardiac myocytes are not well defined. This study examined whether alterations in mitochondrial integrity are involved in TNF-alpha-induced apoptosis in adult ventricular myocytes (ARVM) and determined the roles of caspase-8 (an upstream mediator of TNF-alpha receptor-associated signaling) in this process. After incubation for 24-48 h in serum-free culture medium, ARVM underwent spontaneous apoptosis, which remained stable and was not affected by Z-IETD-FMK, a selective caspase-8 inhibitor. Meanwhile, exposure to TNF-alpha resulted in an increase in apoptosis that was detectable at 6 h and became significant after 12 h, when compared to time-controls. After 24-h exposure, TNF-alpha increased caspase-8 activities, mitochondrial cytochrome C (Cyt C) release to the cytosol, accompanied by loss of mitochondrial transmembrane potential (delta psi(m)). Inhibition of caspase-8 activation in the presence of Z-IETD-FMK abolished the TNF-alpha-induced increases in mitochondrial Cyt C release, loss of delta psi(m) and apoptosis. Therefore, these results suggest that TNF-alpha-induced increase in apoptosis in ARVM results from caspase-8-dependent impairment of mitochondrial integrity.