A colorimetric assay for detection of reverse transcriptase (RT) of the human immunodeficiency virus (HIV) was developed using oligodeoxythymidylic acid (oligo-dT)-linked magnetic beads and digoxigenin-deoxyuridine triphosphate (dig-dUTP). During the RT reaction, dig-dUTP was incorporated into oligo-dT which had been hybridized to polyadenylic acid [poly (A)]. At the detection step, an alkaline phosphatase-conjugated antibody to digoxigenin was added, followed by the addition of a colorimetric substrate for this enzyme. This method showed excellent correlation with the isotopic RT assay, which used tritiated thymidine triphosphate ([3H]dTTP), for detection of purified avian-myeloblastosis-virus RT (AMV-RT). This assay also demonstrated close correlation with the isotopic RT assay using human peripheral-blood lymphocytes infected in vitro with HIV. This colorimetric RT assay offers important advantages over the conventional radioactive RT assays with respect to its simplicity, safety and cost. The total assay time, including the RT reaction step, was less than 1 h, and therefore provides a reliable rapid assay for detection and quantification of HIV.