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To further characterize HIV-1 and HIV-2 Western blot indeterminate (IWB) sera, 402 sera from 318 healthy low-risk individuals from West Virginia and 159 African sera obtained in the pre-AIDS era (1968–1972) were studied. All IWB sera tested for antigen by HIV-1 enzyme immunoassay (EIA-Ag) were negative. HIV-1 and HIV-2 IWB reactivity occurred independent of HIV-1 and HIV-2 false-positive testing for antibody by enzyme immunoassay (EIA-Ab) and no cross-reactions between HIV-1 and HIV-2 IWB patterns were detected. The IWB patterns were reproducible, demonstrated no age or sex related pattern, and showed no evidence of vertical or horizontal transmission. The African sera exhibited a significantly higher number of IWB patterns. IWB reactivity in HIV-1 and HIV-2 seronegative individuals may not be viral in origin and the occurrence of IWB pattern may vary among populations.