Two model substrates were prepared to examine the mechanism of tRNA-primer excision catalyzed by reverse transcriptase associated ribonuclease H (RT-RNase H). The first model substrate contained sequences from the HIV genome and was designed to be structurally similar to the DNA-extended tRNA created by initiation of minus-strand DNA synthesis during retroviral replication. The DNA-extended RNA was a template and was annealed to a DNA oligonucleotide that primed reverse transcription of the RNA in the template. The second model substrate was structurally similar the first substrate but contained sequences unrelated to the HIV viral genome. The RT-RNase H catalyzed excision of the RNA from the template of the two model substrates was examined. Human immunodeficiency virus (HIV) and Moloney murine leukemia virus RT-RNase H hydrolyzed the substrates to leave a single ribonucleotide 5'-phosphate at the 5'-terminus of the model DNA genome. In contrast, avian myeloblastosis virus RT-RNase H hydrolyzed the phosphodiester bond at the DNA-RNA junction. These hydrolytic specificities were not highly dependent on substrate sequence. The importance of these specificities to retroviral integration is discussed. Additional data indicated that the HIV polymerase and RNase H active sites are separated by a distance equivalent to the length of a 15-nucleotide RNA-DNA heteroduplex.