Nuclease footprinting has been used to probe features of binary complexes of type 1 human immunodeficiency virus reverse transcriptase (HIV-1 RT) with both natural and synthetic preparations of its cognate replication primer, tRNA(Lys-3). In addition to heterodimeric RT (p66/p51), ribonucleoprotein complexes containing either the p66 or p51 subunit were analyzed. Footprinting experiments employed both structure- and sequence-specific nucleases. Our results indicate a similar mode of interaction for the three RT preparations tested, suggesting contact with each loop of the tRNA primer (D, anticodon, and T psi C), as well as minor perturbation of the anticodon stem. Although there is little evidence for extensive disruption of the 3'-acceptor stem. RNase A footprinting data with natural and synthetic tRNA suggests that potential base pairing between the T psi C and D loops is disrupted in the presence of RT.