T he intestinal absorption of many nutrients and drug molecules is mediated by ion-driven transport mechanisms in the intestinal enterocyte plasma membrane. Clearly, the establishment and maintenance of the driving forces — transepithelial ion gradients — are vital for maximum nutrient absorption. The purpose of this study was to determine the nature of intracellular pH (pH_i) regulation in response to H⁺-coupled transport at the apical membrane of human intestinal epithelial Caco-2 cells. Using isoform-specific primers, mRNA transcripts of the Na⁺/H⁺ exchangers NHE1, NHE2, and NHE3 were detected by RT-PCR, and identities were confirmed by sequencing. The functional profile of Na⁺/H⁺ exchange was determined by a combination of pH_i, ²²Na⁺ influx, and EIPA inhibition experiments. Functional NHE1 and NHE3 activities were identified at the basolateral and apical membranes, respectively. H⁺/solute-induced acidification (using glycylsarcosine or β-alanine) led to Na⁺-dependent, EIPA-inhibitable pH_i recovery or EIPA-inhibitable ²²Na⁺ influx at the apical membrane only. Selective activation of apical (but not basolateral) Na⁺/H⁺ exchange by H⁺/solute cotransport demonstrates that coordinated activity of H⁺/solute symport with apical Na⁺/H⁺ exchange optimizes the efficient absorption of nutrients and Na⁺, while maintaining pH_i and the ion gradients involved in driving transport.