The sodium−hydrogen exchanger regulatory factor (NHERF) is an essential cofactor for cAMP-mediated inhibition of the Na⁺/H⁺ exchanger isoform, NHE3, in renal brush border membranes. NHERF is also an ezrin-binding protein. To define the functional importance of ezrin binding for NHERF's function as a NHE3 regulator, we transfected stable PS120 cells expressing NHE3 with plasmids encoding WT and truncated mouse NHERF proteins. Co-immunoprecipitation established that in PS120 cells, NHE3 bound to full-length NHERF(1−355), the C-terminal domain, NHERF(147−355), and NHERF(1−325), which lacks the proposed ezrin-binding domain. The N-terminal domain, NHERF(1−146), failed to bind the antiporter. Ezrin was also co-immunoprecipitated with NHERF(1−355) but not with NHERF(1−325). 8Br-cAMP inhibited NHE3 activity in cells that expressed NHERF(1−355) or NHERF(147−355) but had no effect on the formation of NHE3−NHERF or NHERF−ezrin complexes. Na⁺/H⁺ exchange was unaffected by 8Br-cAMP in cells that expressed NHERF(1−146) or NHERF(1−325). NHE3 phosphorylation in vivo was enhanced by 8Br-cAMP only in cells where NHERF bound to both NHE3 and ezrin. The data suggest that NHERF functions as a scaffold to link NHE3 with ezrin and that this multiprotein complex is essential for cAMP-mediated phosphorylation of NHE3 and the inhibition of Na⁺/H⁺ exchange.