Focal adhesion kinase (FAK) has been shown to be activated in cardiac myocytes exposed to mechanical stress. However, details of how mechanical stimuli induce FAK activation are unknown. We investigated whether signaling events mediated by the RhoA/Rho-associated coiled coil-containing kinase (ROCK) pathway are involved in regulation of stretch-induced FAK phosphorylation at Tyr(397) in neonatal rat ventricular myocytes (NRVMs). Immunostaining showed that RhoA localized to regions of myofilaments alternated with phalloidin (actin) staining. The results of coimmunoprecipitation assays indicated that FAK and RhoA are associated in nonstretched NRVMs, but cyclic stretch significantly reduced the amount of RhoA recovered from anti-FAK immunoprecipitates. Cyclic stretch induced rapid and sustained (up to 2 h) increases in phosphorylation of FAK at Tyr(397) and ERK1/2 at Thr(202)/Tyr(204). Blockade of RhoA/ROCK signaling by pharmacological inhibitors of RhoA (Clostridium botulinum C3 exoenzyme) or ROCK (Y-27632, 10 micromol/l, 1 h) markedly attenuated stretch-induced FAK and ERK1/2 phosphorylation. Similar effects were observed in cells treated with the inhibitor of actin polymerization cytochalasin D. Transfection of NRVMs with RhoA antisense oligonucleotide attenuated stretch-induced FAK and ERK1/2 phosphorylation and expression of beta-myosin heavy chain mRNA. Similar results were seen in cells transfected with FAK antisense oligonucleotide. These findings demonstrate that RhoA/ROCK signaling plays a crucial role in stretch-induced FAK phosphorylation, presumably by coordinating upstream events operationally linked to the actin cytoskeleton.