The small G-protein RhoA regulates the actin cytoskeleton and its involvement in cell proliferation has also been established. In contrast little is known about whether RhoA participates in cell survival or apoptosis. In cardiomyocytes in vitro RhoA induces hypertrophic cell growth and gene expression. In vivo, however, RhoA expression leads to development of heart failure (Sah et al JCI, 103, 1999), a condition widely associated with cardiomyocyte apoptosis. We demonstrate here that adenoviral overexpression of activated RhoA in cardiomyocytes induces hypertrophy which transitions over time to apoptosis, as evidenced by caspase activation and nucleosomal DNA fragmentation. The Rho kinase inhibitors Y-27632 and HA-1077, and expression of a dominant negative Rho kinase, block these responses. Caspase-9, but not caspase-8, is activated and its inhibition prevents DNA fragmentation, consistent with involvement of a mitochondrial death pathway. Interestingly RhoA expression induces a 3-4 fold upregulation of the pro-apoptotic Bcl-2 family protein Bax. RhoA also increases levels of activated Bax and the amount of Bax protein localized at mitochondria. Bax mRNA is increased by RhoA, indicating transcriptional regulation, and the ability of a dominant negative (dn) p53 mutant to block Bax upregulation implicates p53 in this response. The involvement of Bax in RhoA-induced apoptosis was examined by treatment with a Bax inhibitory peptide, which was found to significantly attenuate DNA fragmentation and caspase-9 and -3 activation. The dn p53 also prevents RhoA-induced apoptosis. We conclude that RhoA /Rho kinase activation upregulates Bax through p53 to induce a mitochondrial death pathway and cardiomyocyte apoptosis.