The protozoan flagellate Leishmania donovani has an active myo- inositol/proton symporter (MIT), which is driven by a proton gradient across the parasite membrane. We have used site-directed mutagenesis in combination with functional expression of transporter mutants in Xenopus oocytes and overexpression in Leishmania transfectants to investigate the significance of acidic transmembrane residues for proton relay and inositol transport. MIT has only three charged amino acids within predicted transmembrane domains. Two of these residues, Asp19 (TM1) and Glu121 (TM4), appeared to be critical for transport function of MIT, with a reduction of inositol transport to about 2% of wild-type activity when mutated to the uncharged amides D19N or E121Q and 20% (D19E) or 4% (E121D) of wild-type activity for the conservative mutations that retained the charge. Immunofluorescence microscopy of oocyte cryosections showed that MIT mutants were expressed on the oocyte surface at a similar level as MIT wild type, confirming that these mutations affect transport function and do not prevent trafficking of the transporter to the plasma membrane. The proton uncouplers carbonylcyanide-4- (trifluoromethoxy)phenylhydrazone and dinitrophenol inhibited inositol transport by 50-70% in the wild-type as well as in E121Q, despite its reduced transport activity. The mutant D19N, however, was stimulated about 4-fold by either protonophore and 2-fold by cyanide or increase of pH 7.5 to 8.5 but inhibited at pH 6.5. The conservative mutant D19E, in contrast, showed an inhibition profile similar to MIT wild type. We conclude that Asp19 and Glu121 are critical for myo-inositol transport, while the negatively charged carboxylate at Asp19 may be important for proton coupling of MIT.