Cultivation of recently transformed Leishmania mexicana promastigotes at pH 5.5 in Schneider's Drosophila medium supplemented with 20% fetal calf serum produced a homogeneous stationary phase population morphologically similar to metacyclic forms. The cultured forms developed functional characteristics consistent with being metacyclic: they were resistant to complement-mediated lysis, possessed a discernable surface membrane coat in transmission electron micrographs, and were highly infective to peritoneal macrophages in vitro. In contrast, growth of promastigotes at pH 7.0 produced morphologically mixed populations of stationary phase promastigotes, but including a subpopulation with metacyclic-like morphology. These results provide a method for culturing pure populations of L. mexicana metacyclics and provide evidence that metacyclics are biochemically preadapted for survival at acidic pH as amastigotes in macrophage phagolysosomes.