The membrane-coupled exchange of Li+ and/or Na+ for H+ was studied in human platelets measuring intracellular pH(pHi) with a fluorescent indicator. A Li(+)-containing medium restored the internal pH of preacidified platelets to their prior pHi control value. When Na+ was replaced by Li+, similar steady-state values were attained in this system, although it was transported more slowly. The Km and Vmax were both higher with Na+ than with Li+. Exchanges of Li+ or Na+ with H+ were both blocked by ethylisopropylamiloride (EIPA) achieving half-maximal inhibition at submicromolar concentrations. The efflux Li+ or Na+ and resuspended in a choline medium exhibited an influx of H+ sensitive to EIPA. Thrombin, an activator of Na+/H+ exchange, induced a rapid increase in platelet internal pH in the presence of exogenous Li+. Thus: (1) Li+ can be substituted for Na+ in both the forward and the reverse exchange reaction; (2) Li+, while having a higher affinity than Na+ for the external site of the membrane carrier, has a lower Km and (3) Li+ as well as Na+ exchange are activated by thrombin.