The RNase H activity associated with purified avian myeloblastosis virus and Rauscher murine leukemia virus DNA polymerases is inhibited by homopolymeric RNA molecules, although the efficiency of inhibition by each homopolymer appears enzyme specific. Formation of duplex RNA molecules abolished the inhibitory activity. In contrast to these results, DNA polymerase-independent RNase H activities, such as the RNase H-II from Rauscher murine leukemia virus and calf thymus RNase H, were unaffected by the addition of exogenous RNA molecules to reaction mixtures. These results support the concept (M. J. Modak and S. L. Marcus, J. Virol. 22:253--256, 1977) that the catalytic site of DNA polymerase-associated RNase H activity may be that which is also involved in template binding. Naturally occurring RNA molecules of oncornaviral, procaryotic, or eucaryotic origin also proved to be efficient inhibitors of avian myeloblastosis virus DNA polymerase-associated RNase H activity. In contrast to this result, naturally occurring RNA molecules, at concentrations which inhibited the avian myeloblastosis virus enzyme, did not inhibit Rauscher murine leukemia virus DNA polymerase-catalyzed RNase H activity. This finding represents a new biochemical distinction between the two reverse transcriptases, and may be indicative of differences in the relative affinities of these enzymes for natural RNA molecules.