Axenic cultures of Leishmania mexicana amastigotes were transformed to promastigotes in vitro and in vivo in Lutzomyia longipalpis. In vitro, both exponential phase and stationary phase amastigotes were capable of transforming and growing as promastigotes, but exponential phase amastigotes completed this transition more quickly. In vivo, both populations were capable of establishing infections in sand flies by membrane feeding and could be transmitted to BALB/c mice via bite. A variety of morphologic forms could be observed in vivo, including putative metacyclic promastigotes. Infection rates in sandflies with axenic amastigotes were comparable with those achieved with lesion-derived amastigotes, supporting the use of these cultured forms in studies of parasite biology.