A serosurvey of domestic rats was conducted in several South American cities between September 1982 and March 1983 for evidence of hantavirus infection. Antibody-positive rats were found in Belem, São Paulo and Recife-Olinda, Brazil and in Buenos Aires, Argentina, with the highest antibody prevalence rate detected in Belem (30 positive of 54 tested, 56%). A virus isolated from tissues of a Rattus norvegicus captured in Belem, was shown to be antigenically similar to Girard Point viruses isolated from domestic rats captured in the United States and clearly distinct from prototype Hantaan virus, causative agent of Korean hemorrhagic fever in Asia. This represents the first isolation of a virus of the genus Hantavirus from South America and supports previous observations that indicate a widespread distribution of urban rat-associated hantaviruses. The abundance of domestic rats and their regionally high antibody rates suggest that risk of human hantavirus infection in some locations of South America may be significant.