The plasma membrane potential (ΔΨ) of procyclic and bloodstream trypomastigotes of Trypanosoma brucei was studied using the potentiometric fluorescent dye bisoxonol. Our results suggest that a proton pump plays a significant role in the regulation of ΔΨ in procyclic and bloodstream forms, as evidenced by depolarization of the plasma membrane by H -ATPase inhibitors (e.g. dicyclohexylcarbo-diimide, N-ethylmaleimide, diethylstilbestrol, and bafilomycin A₁). In bloodstream stages the plasma membrane was significantly depolarized by ouabain only when the cells were incubated in sodium-rich buffers indicating that a sodium pump was being inhibited. In contrast, ouabain had no effect on the ΔΨ of the procyclic stages in a sodium-rich buffer. However, it induced an additional significant depolarization in these stages when their plasma membrane was already partially depolarized by the H -ATPase inhibitor dicyclohexylcarbo-diimide, indicating the presence of an ouabain-sensitive sodium pump whose activity is masked by the H -ATPase. Unlike procyclic forms, the ΔΨ of bloodstream-stage trypomastigotes was markedly sensitive to extracellular Na and K concentrations. Thus, there are significant differences between procyclic and blooodstream forms in the maintenance of the ΔΨ and in their permeability to cations.