Leishmania major promastigotes are parasites endowed with a plasma membrane electrogenic H+ pump and anionic channels. These systems have been thought to contribute to pH homeostasis of parasites and environmental adaptation by mediating extrusion of protons which are either generated metabolically or result from exogenous acid loads. In this work we show that HCO-3 transport plays a physiological role in supporting pH regulation of parasites. Intracellular pH (pHi) and the membrane potential (Vm) were assessed fluorometrically with pH sensitive and potentiometric dyes. We show that intracellular acidification, caused either by blocking the pump or the putative anion channel or by depleting Cl- from cells, could be largely overcome by addition of HCO-3. Likewise, addition of HCO-3 raises the steady state intracellular pH of untreated cells from 6.76 +/- 0.01 to 6.98 +/- 0.02 and induces membrane hyperpolarization in pump-inhibited cells. We provide evidence for the involvement of HCO-3 transport systems that subserve pH homeostasis in Leishmania promastigotes. A major anionic pathway which is sensitive to anion transport blockers is apparently conductive in nature and accomodates ions such as HCO-3 and Cl-. In physiological conditions, the primary role of H+ pumping is the generation of a relatively large membrane potential (Vm = -113 +/- 4 mV) which subserves electrochemical-driven uptake of nutrients. The involvement of H+ pumping in physiological pH regulation of promastigotes is apparently of a secondary nature.