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This study was conducted to identify the Sarcocystis species that infect the opossum Didelphis aurita in order to determine which sporocysts they are excreating in to the environment and help determine the role of D. aurita in the epidemiology of Sarcocystis. Sporocysts were obtained from intestinal tracts of 8 of 13 D. aurita trapped in Rio de Janeiro state, Brazil, and were orally inoculated into Melopsittacus undulatus and Balb/c nude Mus musculus. Portions of organs and muscles were processed for histology, immunohistochemistry, transmission electron microscopy (TEM), and PCR using primers JNB 33/54, and ITS. Amplification products were subjected to RFLP using DraI and HinfI. Some birds were euthanized 6, 7, 13, 16, and 24 days after inoculation (DAI). All other birds and all mice were euthanized 60 DAI. Schizonts were observed in the lungs using histology and immunostaining in birds examined prior to 60 DAI. Sarcocysts with a ~ 1.5-μm-thick wall were found in the breast, thigh, and tongue of some birds. Sarcocystis asexual stages were isolated in cell cultures inoculated with sporozoites. Parasite DNA isolated from bird tissues and cell cultures demonstrated that S. falcatula-like parasites were present in all samples derived from positive opossums. Asexual stages molecularly characterized as S. lindsayi-like were isolated in cell culture from one opossum with an apparent multiple infection. This study demonstrated that D. aurita is a definitive host for S. falcatula-like parasites and indicates that S. lindsayi-like parasites can be found in coinfections of this opossum species. [ABSTRACT FROM AUTHOR]