pH is one of the most important physiological parameters with its changes affecting the function of vital organs, like the heart. However, the effects of alkalosis on the regulation of cardiac myocyte function have not been extensively investigated. Therefore, we decided to study whether the mitogen-activated protein kinase (MAPK) signaling pathways [c-Jun N-terminal kinases (JNKs), extracellular signal-regulated kinases (ERKs) and p38-MAPK] are activated by alkalosis induced by using the Tris-Tyrode's buffer at two pH values, 8.5 and 9.5, in H9c2 rat cardiac myoblasts. These buffers also induced intracellular alkalinization comparable to the one induced by 1 mM NH₄Cl. The three MAPKs examined presented differential phosphorylation patterns which depended on the severity and the duration of the stimulus. Inhibition of the Na⁺/H⁺ exchanger 1 (NHE1) by its inhibitor HOE642 prevented alkalinization and partially attenuated the alkalosis (pH 8.5)- induced activation of these kinases. The same stimulus also promoted c-Jun phosphorylation and enhanced the binding at oligonucleotides bearing the activator protein-1 (AP-1) consensus sequence, all in a JNKs-dependent manner. Additionally, mitogen- and stress-activated kinase 1 (MSK1) was transiently phosphorylated by alkalosis (pH 8.5) and this was abolished by the selective inhibitors of either p38-MAPK or ERKs pathways. JNKs also mediated Bcl-2 phosphorylation in response to incubation with the alkaline medium (pH 8.5), while selective inhibitors of the three MAPKs diminished cell viability under these conditions. All these data suggest that alkalosis activates MAPKs in H9c2 cells and these kinases, in turn, modify proteins that regulate gene transcription and cell survival.