The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is a ubiquitous plasma membrane protein that regulates intracellular pH by removing a single proton in exchange for one extracellular sodium. The human protein contains a membrane domain of approximately 500 amino acids, and a regulatory, cytosolic domain of approximately 315 amino acids. NHE1 is activated by a number of hormones including endothelin (ET), and by calcium. The regulatory tail possesses an inhibitory calmodulin binding domain and inhibition of NHE1 is relieved by binding of a calcium-calmodulin (CaM) complex. We examined the dynamics of ET-1 and calcium regulation of binding to NHE1 in vivo. Cerulean was linked to the NHE1 protein cytoplasmic C-terminus. This was stably transfected into AP-1 cells that are devoid of their own NHE1 protein. The protein was expressed and targeted properly and retained NHE1 activity comparable to the wild type. We examined the in vivo coupling of NHE1 to CaM by FRET using CaM linked to the fluorescent protein Venus. CaM interaction with NHE1 was dynamic. Removal of serum, reduced CaM interaction with NHE1. Addition of the calcium ionophore ionomycin, increased the interaction between CaM and NHE1. We expressed an ET receptor in AP-1 cells and also found that there was a time dependent association of NHE1 with CaM in vivo that was dependent on ET treatment. The results are the first demonstration of the in vivo association of NHE1 and CaM through ET- dependent signaling pathways.