The variability and discordance of human immunodeficiency virus type 1 (HIV-1) antibody enzyme immunoassay determinations on serial specimens derived, to our knowledge, from the first documented case of HIV-2 infection in North America are described. The initial specimen was weakly reactive, but two subsequent serum specimens were both nonreactive by enzyme immunoassay. All specimens were indeterminate for HIV-1 antibody by HIV-1 Western blot analysis. Serum HIV-2 antibody was demonstrated by enzyme immunoassay using whole virus lysate, HIV-2-specific synthetic peptide assays, and HIV-2 Western blot analysis. Human immunodeficiency virus type 2 genomic sequences were demonstrated in peripheral blood mononuclear cells using gene amplification technology. Human immunodeficiency virus type 2, isolated from peripheral blood lymphocytes, had typical morphologic features of lenti-virus by electron microscopy. Western blot analysis and other specific assays should be considered in individuals with clinical evidence suggesting HIV infection who are nonreactive for HIV-1 antibody by enzyme immunoassay or who have atypical reactivity patterns.