Proline-rich tyrosine kinase 2 (Pyk2) is a nonreceptor protein kinase regulated by intracellular Ca(2+), CaMK, and PKC and can be activated by different stress signals involved in heart failure. However, Pyk2 has not been investigated in the human heart, and the functional role of Pyk2 signaling at the whole heart level has not been elucidated. We hypothesize that Ca(2+)-dependent activation of Pyk2 is involved in cardiac electrophysiology. We examined the expression of Pyk2 in nonfailing versus ischemic and nonischemic failing human hearts (n = 6 hearts/group). To investigate Pyk2 function, we optically mapped perfused hearts from wild-type (WT; n = 7) and knockout (Pyk2(-/-); n = 8) mice during autonomic stimulation. Experiments were done in control mice and after 1 wk of transverse aortic constriction. We used the Illumina beadarray approach for transcriptional profiling of WT and Pyk2(-/-) mouse ventricles. Western blot analysis revealed a doubling of Pyk2 activation in nonischemic failing versus nonfailing human hearts. In mouse hearts, we observed a much higher probability of ventricular tachyarrhythmia during ACh perfusion in Pyk2(-/-) versus WT mice. Parasympathetic stimulation resulted in a dose-dependent decrease of atrial action potential duration (APD) in both WT and Pyk2(-/-) mice, whereas in ventricles it induced APD shortening in Pyk2(-/-) mice but not in WT mice. Deficiency of Pyk2 abolished ACh-induced prolongation of atrioventricular delay in Pyk2(-/-) mouse hearts but did not affect heart rate. Lower mRNA and protein levels of sarco(endo)plasmic reticulum Ca(2+)-ATPase 2 and higher mRNA levels of Na(+)/Ca(2+) exchanger 1 were detected in Pyk2(-/-) hearts compared with WT hearts. The transverse aortic constriction protocol did not change the phenotype. In conclusion, our results indicate a protective role of Pyk2 with respect to ventricular tachyarrhythmia during parasympathetic stimulation by regulation of gene expression related to Ca(2+) handling. We hypothesize that activation of Pyk2 in the human heart during heart failure may contribute to protection against arrhythmia.