Amastigotes of Leishmania mexicana, L. mexicana amazonensis, L. brasiliensis brasiliensis and L. enriettii were isolated from lesions in infected animals [human, Oryzomys capito, guinea pig]. Numbers of amastigotes recovered ranged from 1 .times. 107 to 7 .times. 108, depending on the strain of Leishmania. Trypsinization disassociated the lesions and released the parasites. After 18-24 h incubation at 37.degree. C in tissue culture media with antibiotics, many of the intact host cells attached to the flask. Amastigotes were collected from the media in relatively pure preparations. EM revealed no morphological alterations of the amastigotes and minimal contamination by membranes and cell fragments.