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Signaling events, including Rho GTPases and protein kinase C (PKC), are involved in cardiac hypertrophy. However, the mechanisms by which these pathways cooperate during the hypertrophic process remain unclear. Using an in vitro cyclic stretch model with neonatal rat cardiomyocytes, we demonstrated that stretch‐induced activation of RhoA, Rac1/Cdc42, and phosphorylation of Rho‐guanine nucleotide dissociation inhibitor (GDI) were prevented by inhibition or depletion of PKC, using chelerythrine and phorbol 12‐myristate 13‐acetate, indicating that phorbol ester‐sensitive PKC isozymes may be upstream regulators of Rho GTPases. Using adenoviral‐mediated gene transfer of wild‐type (WT) and dominant‐negative (DN) mutants of PKCα and δ, we found that stretch‐induced activation of Rho GTPases and phosphorylation of Rho‐GDI were mainly regulated by PKCα. PKCδ was involved in regulation of the activation of Rac1. Stretch‐induced increases in [3H]‐leucine incorporation, myofibrillar reorganization and cell size, were blocked by inhibition of Rho GTPases, or overexpression of DN PKCα and δ, suggesting that PKCα and δ are both required in stretch‐induced hypertrophy, through Rho GTPases‐mediated signaling pathways. The mechanism, whereby PKC and Rho GTPases regulate hypertrophy, was associated with mitogen‐activated protein (MAP) kinases. Stretch‐stimulated phosphorylation of MEK1/ERK1/2 and MKK4/JNK was inhibited by overexpression of DN PKCα and δ, and that of MKK3/p38 inhibited by DN PKCδ. The phosphorylation of ERK and JNK induced by overexpression of WT PKCα, and the phosphorylation of p38 induced by WT PKCδ, were regulated by Rho GTPases. This study represents the first evidence that PKCα and δ are important regulators in mediating activation of Rho GTPases and MAP kinases, in the cyclic stretch‐induced hypertrophic process. © 2004 Wiley‐Liss, Inc. [ABSTRACT FROM AUTHOR]