Parallel arrays of Na+/H+and Cl−/HCO3−antiporters are believed to catalyze the first step of transepithelial electrolyte secretion in lacrimal glands by coupling Na+and Cl−influxes across acinar cell basolateral membranes. Tracer uptake methods were used to confirm the presence of Na+/H+antiport activity in membrane vesicles isolated from rabbit lacrimal gland fragments. Outwardly-directed H+gradients accelerated22Na+uptake, and amiloride inhibited 96% of the H+gradient-dependent22Na+flux. Amiloride-sensitive22Na+influx was half-maximal at an extravesicular Na+concentration of 14 mM.In vitrostimulation of isolated lacrimal acini with 10μM carbachol for 30 min increased Na+/H+antiport activity of a subsequently isolated basolateral membrane sample 2.5-fold, but it did not significantly affect Na+/H+antiport activity measured in intracellular membrane samples. The same treatment increased basolateral membrane Na+, K+-ATPase activity 1.4-fold; this increase could be accounted for by decreases in the Na+, K+-ATPase activities of intracellular membranes. Thus, it appears that cholinergic stimulation causes recruitment of additional Na+, K+-ATPase pump units to the acinar cell basolateral plasma membrane. The mechanistic basis of the increase in basolateral membrane Na+/H+antiport activity remains unclear.