Proline-rich tyrosine kinase 2 (PYK2) is a nonreceptor protein tyrosine kinase that links G-protein-coupled receptors to activation of MAPK cascades and cellular growth. In smooth muscle and other cell types, PYK2 activation is dependent on either Ca²⁺ or protein kinase C (PKC), and we have previously shown that endothelin-1 (ET) activates PYK2 in adult and neonatal rat ventricular myocytes (NRVM). However, ET both alters intracellular Ca²⁺ ([Ca²⁺]_i), and activates the novel, Ca²⁺–independent PKCs. Therefore, immunoprecipitation and western blotting experiments were used to examine the PKC and Ca²⁺ dependence of PYK2 activation in NRVM. PYK2 was activated by ET (100 nM; 2–30 min) and phenylephrine (50 μM; 2–30 min), which are both hypertrophic agonists that activate Gq-coupled receptors. Moreover, adenoviral (Adv)–mediated overexpression of constitutively active (ca) Gαq increased PYK2–Y₄₀₂ phosphorylation as early as 8 h post-infection, as compared to NRVM infected with a control Adv encoding β-galactosidase. caGαq overexpression also induced PKCε and PKCδ (but not PKCα) translocation, followed by downregulation of both novel PKC isoenzymes. Phorbol myristate acetate (PMA; 200 nM), a direct activator of Ca²⁺–dependent and Ca²⁺–independent PKCs, activated PYK2 within 10 min, and PYK2 phosphorylation remained elevated after 30 min of stimulation. Adv-mediated overexpression of caPKCε increased PYK2 phosphorylation, whereas Adv-mediated overexpression of a kinase-inactive mutant of PKCε markedly inhibited ET-induced, but not basal PYK2 phosphorylation. In contrast, both basal and ET-induced PYK2 phosphorylation were blocked by treatment with the Src-family protein kinase inhibitor PP2. Although reducing [Ca²⁺]_i with either nifedipine (10 μM) or BAPTA-AM (50 μM) decreased basal PYK2 phosphorylation, it did not prevent ET-induced PYK2 activation. Furthermore, increasing [Ca²⁺]_i with ionomycin (10 μM), K⁺ depolarization, or BayK8644 (1 μM) was not sufficient to further activate PYK2. These data demonstrate that ET-induced PYK2 activation is Gq, PKCε, and Src dependent, describing a distinct signaling pathway leading to agonist-induced PYK2 activation in cardiomyocytes.