A highly sensitive nested polymerase chain reaction (PCR) protocol was used to detect human immunodeficiency virus type 1 (HIV-1) DNA in peripheral blood mononuclear cells from 271 HIV-1-seropositive patients, 240 HIV-1-seronegative subjects at increased risk for HIV-1 infection, 51 serologically indeterminate individuals, and 120 healthy blood donors. PCR was carried out in a multiplex nested configuration with pol and env region primer sets. HIV-1 DNA was detected in all of the HIV-1 seropositive patients. In contrast, HIV-1 DNA was not detected in any of the either seronegative or serologically indeterminate subjects. Only one of 37 seronegative regular sexual partners of HIV-1-infected patients who were followed longitudinally was found to seroconvert to HIV-1. However, HIV-1 DNA and antibody results were concordant in the four samples obtained from this subject prior to and after seroconversion. These results show an excellent concordance between HIV-1 DNA and antibody detection for diagnosis of HIV-1 infection and suggest that long-term HIV-1 infection in the absence of detectable antibody is likely to occur at a very low frequency.