The polymerase chain reaction (PCR) fails to detect HIV-1 sequences in 5% of infected individuals. To screen for false-negative PCR tests, we developed a nonisotopic PCR assay in which sequences from the beta-globin gene and from the HIV-1 vpu-env region were coamplified with biotinylated and fluorescein-labeled primers, respectively. Coamplified products were reacted with specific internal digoxigenin-labeled RNA probes. Hybrids were detected in a microtiter plate coated with streptavidin or anti-fluorescein antibody, with enzyme-labeled anti-digoxigenin antibody. After the optimization of the coamplification conditions, the assay could detect 5 proviral DNA copies in a lysate from 100,000 peripheral blood mononuclear cells. Fifty-seven samples from 55 HIV-1-seropositive patients and 25 samples from 25 seronegative individuals were evaluated. Fifty-two samples from HIV-infected individuals were positive for HIV-1 vpu-env sequences. Three of the 5 PBMC lysates falsely negative for HIV-1 sequences had reactivities for beta-globin (3-23 fu) below that of 100,000 cells (304 fu). Nonisotopic coamplification allowed for the evaluation of the quality of specimens for PCR concurrently with the detection of the presence of viral template sequences.