As a first step toward identifying putative regulators of apoptosis in the heart, the impact of the anti-apoptosis protein Bcl-2 (B-cell lymphoma gene) on the NFκB (nuclear factor kappa beta) signalling pathway in suppressing apoptosis in ventricular myocytes was studied. The data indicate that adenovirus-mediated delivery of Bcl-2 resulted in a significant increase in NFκB-dependent DNA binding and NFκB-directed gene transcription. No change in NFκB protein content was observed in myocytes expressing Bcl-2. Moreover, the Bcl-2-mediated NFκB activation was found to be related to changes in the activity of the NFκB regulatory protein IκBα (inhibitor of kappa beta). In this regard, a marked reduction in IκBα protein content was observed in ventricular myocytes expressing Bcl-2. The mode by which Bcl-2 regulates IκBα was related to the N-terminal phosphorylation and degradation of IκBα by the proteasome since an N-terminal deletion mutant of IκBα or the proteasome inhibitor lactacystin abrogated Bcl-2’s inhibitory effects on IκBα and prevented NFκB activation. Furthermore, adenovirus-mediated delivery of a phosphorylation defective form of IκBα rendered ventricular myocytes incapable of NFκB activation and susceptible to tumour necrosis factor alpha-mediated apoptosis. Moreover, Bcl-2’s anti-apoptotic function was lost in cells defective for NFκB activation. The data provide evidence for a link between Bcl-2 and the NFκB signalling pathway for the suppression of apoptosis in ventricular myocytes.