To examine the effects of increased expression of connexin43 (Cx43) upon cell viability and response to cytotoxic agents, we expressed Cx43 in LNCaP and PC3 prostate cancer cells by infection with a recombinant adenovirus (Ad-Cx43). Infection with Ad-Cx43 led to the formation of Cx43-containing gap junction plaques at appositional membranes and increased Lucifer Yellow transfer in LNCaP cells, but not in PC3 cells. The increased intercellular communication was blocked by co-infection with an adenovirus containing a dominant-negative Cx43 (Ad-Cx43DN). Infection of LNCaP (but not PC3) cells with Ad-Cx43 greatly increased their sensitivity to killing by tumor necrosis factor alpha (TNFalpha), anti-Fas antibodies, and TRAIL as quantified using an MTS assay. The TNFalpha-induced cell death was dependent on cell density, and it was associated with increased annexin V staining, an increased proportion of sub-G1 cells, and activation of caspase 8. The TNFalpha-induced effects on Ad-Cx43-infected LNCaP cells were blocked by co-infection with Ad-Cx43DN or by pre-incubation with neutralizing antibodies directed against TNFalpha receptor 1. These results demonstrate that TNFalpha induces apoptosis in LNCaP cells by signaling through TNFalpha receptor 1 and that expression of functional Cx43 gap junction channels increases their sensitivity to TNFalpha.