The NHE2 isoform of the Na+/H+ exchanger (NHE) displays two proline-rich sequences in its C-terminal region that resemble SH3 (Src homology 3)-binding domains. We investigated whether these regions (743PPSVTPAP750, termed Pro-1, and 786VPPKPPP792, termed Pro-2) can bind to SH3 domains and whether they are essential for NHE2 function and targeting. A fusion protein containing the Pro-1 region showed promiscuous binding to SH3 domains of several proteins in vitro, whereas a Pro-2 fusion bound preferentially to domains derived from kinases. In contrast, cytoplasmic regions of NHE1, NHE3, or NHE4 failed to interact. When expressed in antiporter-deficient cells, truncated NHE2 lacking both Pro-rich regions catalyzed Na+/H+ exchange, retained sensitivity to intracellular ATP, and was activated by hyperosmolarity, resembling full-length NHE2. The role of the Pro-rich regions in subcellular targeting was examined by transfection of epitope-tagged forms of NHE2 in porcine renal epithelial LLC-PK1 cells. Both full-length and Pro-2-truncated NHE2 localized almost exclusively to the apical membrane. By contrast, a mutant devoid of both Pro-1 and Pro-2 was preferentially sorted to the basolateral surface but also accumulated intracellularly. These observations indicate that the region encompassing Pro-1 is essential for appropriate subcellular targeting of NHE2.