We examined the ribonuclease H (RNase H) specificity of human immunodeficiency virus reverse transcriptase (HIV-RT) using heteropolymeric RNAs hybridized to complementary DNAs. Experiments were performed in the presence of excess challenger polymer (poly(rA)-oligo(dT)) to reveal cleavages resulting from single enzyme binding events. Previous results suggested that initial RNase H directed cleavages were a fixed distance from a DNA primer terminus recessed on an RNA template, i.e. determined by the binding position of the polymerase active site. The influences of recessed RNA termini were not evaluated. In current experiments, RNAs that were 30, 42, or 50 nucleotides long were hybridized to the same 88 nucleotide long complementary DNA, such that the 5' terminal nucleotide of each RNA was hybridized to the 29th nucleotide from the 3' end of the DNA. In all three cases the RNA was initially cleaved between the 19th and 21st nucleotides from its 5' end. Thus, cleavage was not coordinated by the recessed 3' terminus of the RNA. Subsequent cleavages in either direction on the RNA were also observed. An insertion within the RNA that moved the preferred initial cut sequence 10 nucleotides further from the 5' end of the RNA decreased but did not abolish cleavage at the sequence. However, changing the nucleotide sequence in the region of the preferred cleavage either by the insertion experiment or mutagenesis did not significantly alter its capacity for cleavage. These results demonstrated a dominant position preference, plus a sequence priority. In another experiment, a 25 nucleotide long DNA was hybridized such that its 3' terminal nucleotide was 9 nucleotides from the 5' end of a 60 nucleotide complementary RNA. The preferred RNA cleavage sequence discussed above, was 10-14 nucleotides upstream of the 3' end of the DNA. However, initial cleavages occurred 17-20 nucleotides from the DNA 3' end, consistent with cleavage being coordinated by the recessed 3' terminus of the DNA primer.