The 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 phi and the ion gradients involved in driving transport.