Leishmania protozoans are the causative agents of leishmaniasis, a major parasitic disease in humans. The parasites manifest a nutritional requirement for heme compounds since they are deficient in heme biosynthesis. In this study we have demonstrated for the first time the presence of the enzyme L-alanine: 4,5-dioxovalerate transaminase in Leishmania donovani. This enzyme catalyzes the synthesis of 5-aminolevulinic acid (ALA), the first committed step in heme synthesis. Thus the defect in heme biosynthesis pathway in Leishmania must lie at some enzymatic level subsequent to synthesis of ALA. The enzyme was found to be present in both virulent and avirulent strains of L. donovani. The virulent promastigotes showed a 41% higher specific activity as compared to the avirulent strain. The enzyme activity was found to be inhibited in the presence of heme and methylglyoxal. Immunoblot analysis revealed that L-alanine: 4,5-dioxovalerate transaminase in L. donovani was immunologically different from that in mammals.