A sensitive biochemical assay of viral reverse transcriptase (RT) was developed that is useful for both the detection and quantitation of human immunodeficiency virus (HIV), the agent responsible for acquired immune deficiency syndrome in humans. This assay gave a 20- to 40-fold increase in enzyme activity over the current method used for RT detection of HIV. The test is based on a previous biochemical study showing the unusual stability of avian oncornavirus RNA-dependent DNA polymerases at 30 degrees C for at least 2 days. Our study shows that the HIV polymerase is stable at 30 to 37 degrees C for up to 3 days. By using this sensitive RT assay, as few as 250 HIV virions can be quantitated directly in tissue culture medium. This assay should prove useful in studies in which the detection of HIV or the quantitation of the number of virions is required.