The symptoms of hantavirus pulmonary syndrome may resemble those of other febrile illnesses. The development of an accurate diagnostic test should therefore improve clinical prognosis and be useful in epidemiologic studies. We evaluated the use of a recombinant antigen (rNDelta(85)) based on the S-segment sequences of a Brazilian hantavirus for detecting immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies against hantavirus in an indirect enzyme immunoassay (EIA). We assayed 613 serum samples (570 from humans and 43 from rodents). IgM EIA had a sensitivity of 94.1% and a specificity of 99.1%. IgG EIA had a sensitivity of 95.2% and a specificity of 98.4%. This evaluation confirms that rNDelta(85) IgM and IgG EIA tests are potentially useful rapid, sensitive, and cost-effective tools for detecting antibodies against hantaviruses indigenous to Brazil and other South American countries, in patients with acute or convalescent hantavirus infection, and in rodent reservoirs.