Chagas disease, a zoonotic disease caused by the bloodborne parasite Trypanosoma cruzi, affects an estimated 11 million persons throughout much of Latin America. In endemic areas, T. cruzi is transmitted primarily by triatomine insects (i.e., kissing bugs); infection also can occur via blood transfusion, congenital transmission, organ transplantation, laboratory incident, and ingestion of triatomine-contaminated food or drink. To evaluate an investigational assay for detecting T. cruzi infection in blood donations, the American Red Cross conducted a clinical trial during August 2006-January 2007, screening 148,969 blood samples at three blood-collection centers in the United States. In January 2007, after the new assay was licensed by the Food and Drug Administration (FDA), other centers began screening donors for T. cruzi. This report describes the results of the American Red Cross study, which identified 32 donations (approximately one in 4,655) as confirmed positive for T. cruzi antibodies. As blood-donation screening for Chagas disease becomes more widespread, public health officials and health-care providers should anticipate increased numbers of questions regarding the diagnosis, evaluation, and management of Chagas disease.