The first step in the formation of lacrimal gland fluid is believed to depend on transport systems which couple a flux of Cl- ions to the passive influx of Na+ ions across the acinar cell basal-lateral plasma membrane. The transport systems which mediate these fluxes have not yet been characterized, but a review of previous studies (Parod and Putney, Am J Physiol 239:G106, 1980) raises the possibility that Na+/H+ antiporters might represent a major pathway for Na+ influx. This conclusion is of interest, because antiporter mediated Na+ fluxes can, potentially, drive net Cl- fluxes. We have now examined a sample of basal-lateral membrane vesicles from rat exorbital lacrimal gland to verify the presence of a Na+/H+ antiporter activity. Imposition of an outward H+ gradient caused a 4.4-fold increase in the 22Na influx rate, while imposition of an outward Na+ gradient accelerated H+ uptake as determined by changes in acridine orange absorbance. All transport experiments were done in the presence of valinomycin and symmetrical K+ concentrations, eliminating the possibility of conductive Na+ or H+ fluxes driven by diffusion potentials. The pH gradient dependent Na+ influx was completely inhibited by 1 mM amiloride, indicating that it was mediated by a Na+/H+ antiporter similar to those described in other tissues. Comparison of the density distributions of Na+/H+ antiport and standard membrane marker enzyme activities confirmed that the antiporter was primarily localized to the basal-lateral membranes.