Three methods were used to measure intracellular pH (pHi) of Leishmania donovani promastigotes: (a) measurement of the fluorescence of the pH indicator 2',7'-bis-(carboxyethyl)-5,6-carboxyfluorescein; (b) pH null point assays; and (c) determination of the distribution across the promastigote plasma membrane of the fluorescent amine acridine orange and of the weak acid 5,5-dimethyl-2,4-oxazolidinedione. The three methods gave similar results and showed that promastigotes of L. donovani maintain pHi at a narrow range of 6.4-6.7, throughout an extracellular pH (pHo) range of 5.5-7.4. L-Proline transport in L. donovani promastigotes, which is known to be coupled to proton translocation, was used to estimate the proton electrochemical gradient across parasite plasma membrane. While proline uptake is optimal at pHo 7.5, an outward-directed concentration gradient is obtained at steady state throughout a pHo range of 5-8. The calculated electrochemical gradient of proline across the parasite plasma membrane at steady state is 90-100 mV within a pHo range of 5-8, suggesting an almost constant proton electrochemical gradient at this pHo range. Taken together, the results show that the parasites regulate both pHi and the size of the chemiosmotic energy required to drive active transport of nutrients.