Regulation of renal Na-HCO cotransporter (NBC1) activity by cholinergic agonists, ANG II, and acute acidosis (CO₂) requires both Src family kinase (SFK) and classic MAPK pathway activation. The nonreceptor tyrosine kinase proline-rich tyrosine kinase 2 (Pyk2) couples discrete G protein-coupled receptor and growth factor receptor signaling to SFK activation. We examined the role of Pyk2-SFK interaction in coupling these stimuli to increased NBC1 activity in opossum kidney cells. Carbachol increased tyrosine autophosphorylation of endogenous Pyk2 and ectopically expressed wild-type Pyk2 and were abrogated by kinase-dead mutant (Pyk2-KD) overexpression. Pyk2 phosphorylation was calcium/calmodulin dependent, and Pyk2 associated with Src by means of SH2 domain interaction. Pyk2 phosphorylation and Pyk2-Src interaction by carbachol were mimicked by both ANG II and CO₂. To correlate Pyk2 autophosphorylation and Pyk2-Src interaction with NBC activity, cotransporter activity was measured in untransfected cells and in cells overexpressing Pyk2-KD in the presence or absence of carbachol, ANG II, or CO₂. In Pyk2-KD-overexpressing cells, the effect of carbachol, ANG II, and CO₂ was abolished. We conclude that Pyk2 plays a central role in coupling carbachol, ANG II, and CO₂ to increased NBC activity. This coupling is mediated by Pyk2 autophosphorylation and Pyk2-Src interaction.