Endocytosed ligands move through a series of progressively more acidic vesicles. These differences in pH (pHi) could reflect differences in ion transport mechanisms. Vesicles representing three stages of endocytosis, compartment for uncoupling of receptor and ligand (CURL), multivesicular bodies (MVB), and receptor recycling compartment (RRC), were studied, and all exhibited ATP-dependent electrogenic acidification that was a saturable function of medium chloride. Initial rates of acidification differed (RRC > CURL > MVB), and proton influx was similar for CURL and RRC but slower for MVB. Steady-state ATP-dependent pHi in the three vesicles was more similar. Vesicle membrane potential was substantial (+41 to +69 mV) in low-chloride medium and greatest for RRC but was low (-6 to +6 mV) in 140 mM KCl. These vesicles also exhibited -22 to -37 mV Donnan potentials. Steady-state pump-generated proton electrochemical gradients (delta mu H+) ranged from 114 to 175 mV and were greater for CURL and RRC than for MVB; however, delta mu H+ changed little over a 140-fold difference in chloride concentration. Proton leak rates were faster in CURL and RRC than in MVB, but proton efflux was similar. Finally, proton fluxes and permeabilities, calculated with regard to surface area, differed in the opposite direction (MVB > CURL > RRC). Thus, for the endocytic vesicles studied, intrinsic differences in proton flux and in vesicle geometry could be demonstrated that contributed to differences in pre-steady-state vesicle pHi.