We have previously shown that Leishmania donovani promastigotes adapted to long-term culture at acidic pH can serve as a model to study parasite development in a lysosomal-like environment. In this study we investigated the effect of growth pH on L. donovani L-proline transport systems. Reducing the pH of the growth medium causes an up to 7-fold decrease in the extent of L-proline transport. Transport resumes after switching the culture from pH 4.5 to pH 7 for 48 h by a protein synthesis-dependent process. The pH optimum for transport changes from 7.5 in promastigotes grown at pH 7 to 5.5 in cells grown at pH 4.5. In addition, kinetic analysis of L-proline transport showed that Vmax in pH 4.5-grown L. donovani promastigotes is one-tenth that of cells grown at pH 7 (4.5 and 44.7 nmol min-1 (10(8) cells)-1, respectively). The apparent Km for L-proline in pH 4.5 promastigotes is one-half of the Km in pH 7 cells (0.30 and 0.65 mM, respectively). In contrast to L-proline transport, D-glucose transport demonstrates a growth pH-independent activity: Km and Vmax as well as optimum pH of transport are similar in promastigotes grown at either pH 7 or pH 4.5. Taken together, the results indicate that in L. donovani, expression and activity of L-proline transport is regulated by culture pH. The pH-dependent expression of L-proline transporters may be of physiological significance during the promastigote-amastigote transition.