Amastigotes of Leishmania mexicana pifanoi were cultivated by serial transfers in cell-free medium UM-54 at 33 and 35 C. Electron microscopy was used to analyze the structural relationships among promastigotes, axenically cultured amastigotes, and amastigotes in footpads of infected hamsters. These studies revealed very close structural similarities between culture and hamster derived amastigotes. However, both of these amastigotes differed from the promastigotes in the following aspects. The flagellum of promastigotes contained a paraxial rod originating at the axosome level within the flagellar pocket, whereas the flagellum of amastigotes lacks this structure. The flagellar pocket of promastigotes was usually small whereas amastigotes had a distended reservoir. Subpellicular microtubules of promastigotes terminated at the posterior end, whereas those of amastigotes ended subterminally. Membrane bounded vesicles were present only in amastigotes. These results along with the biologic and antigenic comparisons indicate that amastigotes obtained from axenic cultures are related very closely to amastigotes from infected hamster footpads and that their relationship to promastigotes is far more distant.