Thirty-five specimens of Philander frenata and 36 Didelphis marsupialis were captured in the same Atlantic forest area of Brazil between 1992 and 1994. Haemocultures showed that 50% of P. frenata and 60% of D. marsupialis were infected with Trypansoma cruzi. Biological, biochemical and molecular characterization of the isolates suggested 2 distinct transmission cycles of T. cruzi occurred between these 2 sympatric didelphids. The T. cruzi isolates could be distinguished according to their association with each marsupial species. Biochemical characterization (multilocus enzyme electrophoresis) revealed 15 zymodemes; more variability was observed among the P. frenata isolates than among the isolates from D. marsupialis. The course of natural and experimental infection in D. marsupialis and P. frenata was different and suggested that D. marsupialis was more resistant to infection than P. frenata. In the studied area, P. frenata seems to be a more important reservoir of T. cruzi than D. marsupialis, since 40% of the characterized isolates from P. frenata belonged to the T. cruzi II group, which is associated with human infections.