Metabolism of tryptophan by promastigotes of Leishmania donovani donovani was investigated in cells suspended in a simple buffer solution supplemented with glucose. Metabolites from supernatant and lysed cell pellets were analyzed by capillary gas liquid chromatography and 13C nuclear magnetic resonance spectroscopy, with structural confirmation by gas liquid chromatography-mass spectrometry. Tryptophan does not appear to serve as a carbon energy source for L. d. donovani promastigotes since parasites could survive for only short periods in buffer containing tryptophan without glucose, levels of tricarboxylic acid cycle intermediates remained unchanged in the presence of added tryptophan and label from [13C]tryptophan was not detected in any of the intermediates. Leishmania d. donovani catabolized L-tryptophan via aminotransferase and aromatic lactate dehydrogenase reactions to form one major end product, indole-3-lactic acid. The activity of aromatic lactate dehydrogenase required manganese and was NADH-dependent in these organisms that lack lactate dehydrogenase. Promastigotes taken from the mid-log stage of growth produced higher concentrations of indole-3-lactic acid than those from the stationary stage. Conservation of a similar tryptophan catabolic pathway among four Leishmania species suggests the pathway is physiologically important to the parasites themselves.