Trypanosoma (Megatrypanum) freitasi, a parasite of didelphid opossum, was known to be very difficult to cultivate in conventional media. Co-cultivation with L929 cell line in Baltz's medium at 27.5 degrees C resulted in luxuriant growth of the trypanosome with the production of epimastigote colonies that adhered to the surface of culture flasks or tubes, and transformation into metacyclics. Further transformation was stimulated by raising the incubation temperature. At 37 degrees C the population was of the bloodstream type and resistant to lysis by complement.