The type 1 sodium-proton exchanger (NHE-1) is expressed ubiquitously and regulates key cellular functions, including mitogenesis, cell volume, and intracellular pH. Despite its importance, the signaling pathways that regulate NHE-1 remain incompletely defined. In this work, we present evidence that stimulation of the 5-hydroxytryptamine 1A (5-HT1A) receptor results in the formation of a signaling complex that includes activated Janus kinase 2 (Jak2), Ca2+/calmodulin (CaM), and NHE-1, and which involves tyrosine phosphorylation of CaM. The signaling pathway also involves rapid agonist-induced association of CaM and NHE-1 as assessed by coimmunoprecipitation studies and by bioluminescence resonance energy transfer studies in living cells. We propose that NHE-1 is activated through this pathway: 5-HT1A receptor --> G(i2)alpha and/or G(i3)alpha --> Jak2 activation --> tyrosine phosphorylation of CaM --> increased binding of CaM to NHE-1 --> induction of a conformational change in NHE-1 that unmasks an obscured proton-sensing and/or proton-transporting region of NHE-1 --> activation of NHE-1. The G(i/o)-coupled 5-HT1A receptor now joins a handful of Gq-coupled receptors and hypertonic shock as upstream activators of this emerging pathway. In the course of this work, we have presented clear evidence that CaM can be activated through tyrosine phosphorylation in the absence of a significant role for elevated intracellular Ca2+. We have also shown for the first time that the association of CaM with NHE-1 in living cells is a dynamic process.