Cultures of Trypanosoma ambystomae were initiated and maintained in hypo-osmotic biphasic blood-agar medium. Epimastigotes and trypomastigotes occurred in culture and although they were similar in size, epimastigotes predominated with about 70% of all stages. Epimastigotes divided by equal binary fission and occasionally formed rosettes. Cultured trypomastigotes did not divide. Cultures isolated 118 days earlier were infective to all 3 laboratory-reared Ambystoma jeffersonianum and to 2 of 3 Ambystoma mexicanum; the latter is a new host record. Trypanosomes were morphometrically similar in both experimental hosts. Specimens from 20-day-old experimental infections were smaller than those from natural infections. Dividing bloodstream forms were detected in experimental infections. Standard culture conditions should be adopted if the morphology of culture forms is to have taxonomic significance.