Leishmaniasis is a neglected tropical disease that continues to pose a public health threat worldwide due to the absence of an effective vaccine, drug toxicity and parasite resistance. In an attempt to identify new potential drug targets, we focused our research on Leishmania donovani argininosuccinate synthase (LdASS), which is more highly expressed in the virulent form of the parasite. Using two cell lines that over expressed the wild type or a mutant form of LdASS, we demonstrated that LdASS has argininosuccinate synthase activity, which is absent in the mutant form containing the G128S point mutation. Infection of mice with the cell line over expressing a mutant LdASS had a negative dominant effect as indicated by the reduction in parasite load. LdASS is localized to large cytosolic complexes and a small portion is in a new vesicular subset different from the known glycosomes. Thus LdASS constitutes a new virulence factor that may be a potential drug target.