Nutrient broth containing fetal calf serum was used successfully in isolating Leishmania donovani from animals and cryopreserving Leishmania major, Leishmania donovani, and Leishmania adleri. It also supported heavy growth of promastigotes of laboratory strains of L. donovani, L. major, L. adleri, and uncharacterized reptilian Leishmania-like flagellates.