Many hypertrophic stimuli such as angiotensin II (Ang II) activate phospholipases through G protein–coupled receptors in cardiac myocytes. However, it is not known whether these stimuli also activate the tyrosine phosphorylation–dependent signaling pathway, which plays an essential role in growth factor–induced mitogenic responses in other cell types. Serine/threonine kinases such as mitogen-activated protein (MAP) kinases and 90-kD S6 kinase (RSK) are activated in response to many growth stimuli and are important downstream signaling pathways of tyrosine kinases. Therefore, we examined whether Ang II activates these protein kinases in primary cultures of cardiac myocytes and fibroblasts from neonatal rats. Ang II rapidly induced tyrosine phosphorylation of multiple proteins, including 42-, 44-, 75- to 80-, and 120- to 130-kD proteins, in both cardiac myocytes and fibroblasts. This was accompanied by an increase in tyrosine kinase activity. The 42- and 44-kD proteins were immunologically related to an extracellular signal-regulated kinase family (MAP kinases). Ang II rapidly increased kinase activity of MAP kinases and their downstream kinase, RSK. The Ang II–induced tyrosine phosphorylation and activation of MAP kinases and RSK were AT₁ receptor–mediated. Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate or an increase in intracellular Ca²⁺ by the Ca²⁺ ionophore A23187 was sufficient to cause tyrosine phosphorylation of multiple proteins and activation of MAP kinase and RSK. Although downregulation of PKC did not suppress Ang II–induced activation of MAP kinase and RSK, chelating intracellular Ca²⁺ by BAPTA-AM completely abolished Ang II–induced activation of these kinases. Activation of MAP kinases and RSK was also observed in myocytes stimulated with other agonists for G_q protein–coupled receptors, such as phenylephrine, norepinephrine, and endothelin 1, but not with agonists to G_s protein–coupled receptors, such as isoproterenol. These results suggest that Ang II and other hypertrophic stimuli, known to act through G_q protein–coupled receptors, rapidly cause tyrosine phosphorylation of several intracellular substrates through activation of tyrosine kinase and activate MAP kinases and RSK in cardiac myocytes as well as in cardiac fibroblasts. Furthermore, intracellular Ca²⁺, rather than PKC, seems to be critical for Ang II–induced activation of these protein kinases in cardiac myocytes.