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ABSTRACT The protozoan parasite Sarcocystis falcatula is an important cause of clinical disease in several avian intermediate hosts. The host range of S. falcatula is wide, and numerous outbreaks of acute sarcocystosis have been reported in passerine and psittacine birds in captivity in the Americas. Previous diagnosis was performed by serologic methods, light, and/or electron microscopic examinations with limited molecular confirmation. Here, we report histological and molecular diagnosis of acute, fatal S. falcatula infections in rainbow lorikeets (Trichoglossus moluccanus) at the Philadelphia Zoo. Pulmonary sarcocystosis was suspected antemortem in 3 lorikeets (3–5 yr old); these birds died despite antiprotozoal therapy. The predominant lesion was pneumonia associated with S. falcatula–like schizonts in pulmonary vascular endothelium. The multilocus PCR-DNA sequencing (18S rDNA, 28S rDNA, ITS-1, and cox1) of frozen lung tissue confirmed S. falcatula infections in all 3 birds. Our results and previous studies suggest that acute pulmonary form of sarcocystosis is a major contributor of death to Old World psittacine birds.