Recently, we demonstrated that pulsatile mechanical stretch induced rapid secretion of vascular endothelial growth factor (VEGF) by cultured rat cardiac myocytesin vitro.To investigate whether pulsatile stretch activates intracellular signaling in cardiac myocytes, we examined the activation of mitogen-activated protein kinase (MAPK) family members and focal adhesion kinase (p125^FAK) in cultured rat cardiac myocytes. We found that pulsatile stretch rapidly phosphorylated p44/p42 MAPKs (extracellular signal-regulated protein kinase [ERK] 1/2), stress-activated protein kinase (SAPK), p38MAPK, and p125^FAK. The stretch-induced activation of ERKs was at least partly mediated by VEGF, which was shown to be induced by transforming growth factor (TGF)-β, and was also partly dependent on tyrosine kinases as well as protein kinase C (PKC). These data provide the direct evidence that pulsatile stretch can activate intracellular signaling in cardiac myocytes and that this was at least partly mediated by VEGF, which may play a role in cardiac adaptation to mechanical overload.