The aim of this study was to detect HIV-1 proviral DNA in lysates of peripheral blood mononuclear cells (PBMCs) by the polymerase chain reaction (PCR) and hybridization with a nonradioactive probe. PBMCs were lysed in 1% Triton X-100. PCR was then carried out using primers complementary to a conserved region of the HIV-1 pol gene. Bracket and nested amplification protocols were used. Products were identified by dot-blot hybridization or agarose gel electrophoresis and Southern hybridization, using an alkaline phosphatase-linked oligonucleotide probe specific for amplified sequences. Colorimetric and chemiluminescent substrates were used. HIV-1 DNA was detected in PBMCs of 57/59 HIV-1-seropositive individuals, 8 of which were positive only following the use of nested primers. Of 12 seropositive samples that were negative by other HIV-1 diagnostic tests (PBMC coculture and serum p24 antigen detection), 11 were positive by PCR. PCR using PBMC lysates is a very sensitive method of detecting HIV-1 proviral sequences. The use of nested primers appears to increase the sensitivity of the procedure.