Human immunodeficiency virus reverse transcriptase (HIV-RT) binds more stably in binary complexes with RNA-DNA versus DNA-DNA. Current results indicate that only the -2 and -4 RNA nucleotides (-1 hybridized to the 3´ recessed DNA base) are required for stable binding to RNA-DNA, and even a single RNA nucleotide conferred significantly greater stability than DNA-DNA. Replacing 2´- hydroxyls on pivotal RNA bases with 2´-O-methyls did not affect stability, indicating that interactions between hydroxyls and RT amino acids do not stabilize binding. Avian myeloblastosis and Moloney murine leukemia virus RTs also bound more stably to RNA-DNA, but the difference was less pronounced than with HIV-RT. We propose that the H- versus B-form structures of RNA-DNA and DNA-DNA, respectively, allow the former to conform more easily to HIV-RT's binding cleft, leading to more stable binding. Biologically, this may aid in degradation of RNA fragments that remain after DNA synthesis.