A hybridization protection assay (HPA) that uses acridinium ester (AE) labeled oligonucleotide probes which are specific for a conserved gag gene region of human immunodeficiency virus type 1 (HIV-1) was developed to measure the amount of HIV-1 nucleic acid. Hybridization of the single-stranded probes with their target HIV-1 sequences protected the chemiluminescent AE group from subsequent alkaline hydrolysis. The chemiluminescence from the residual AE could be easily quantitated in a luminometer. The entire process comprising template dissociation, hybridization, alkaline hydrolysis, and chemiluminescence measurement can be completed in less than one hour and does not require the separation of hybridized probe from unhybridized probe. We demonstrated that HPA could quantitatively measure the amount of DNA amplified by polymerase chain reaction. A comparative study using amplified DNA from the peripheral blood mononuclear cells (PBMC) of HIV seropositive and seronegative persons showed that HPA was as sensitive as the previous methods using 32P-labeled DNA probes.