6-(P-hydroxyphenylazo)-uracil (HPUra) reduced by dithiothreitol inhibited AMV or RLV virion associated exogenous RNA-dependent DNA polymerase reactions. However, the inhibition was variable from experiment to experiment and was not consistent with the base specificity of HPUra seen for inhibition of gram positive DNA-dependent DNA polymerases. Increasing the concentration of dithiothreitol reversed the inhibition. Furthermore, at non-toxic concentrations, HPUra did not influence the plating efficiency of RLV in tissue culture, as measured by the ability to induce foci on sarcoma virus positive-leukemia virus negative cells. Oxidation of dithiothreitol in the presence or absence of HPUra was followed spectrophotometrically under enzyme conditions. HPUra catalyzed the oxidation of dithiothreitol under these conditions. Since dithiothreitol is required for optimum reaction rates, as well as complete disruption of virus in some polymerase assay systems, the oxidation of dithiothreitol in the presence of HPUra is sufficient to explain the inhibition seen.