The transcription initiation primer for HIV-1 is a specific cellular tRNA species, tRNA Lys3 . We used several methods to assess the binding of tRNA by recombinant HIV-1 p51/p66 reverse transcriptase (RT), gel retardation analysis, intrinsic RT protein fluorescence quenching, and nitrocellulose filter binding assays. The binding of tRNA to RT was saturable, implying a distinct site or sites on the enzyme for tRNA interaction. However, this binding was non-selective, with all tRNA isoacceptors and total unfractionated tRNA binding with similar affinity as primer tRNA Lys3 . In contrast, no significant binding of rRNA by RT was noted. Our results show that HIV-1 RT has no specificity for the binding of primer tRNA Lys3 , and imply that factors other than RT sequences may be important for the selective incorporation of primer tRNA into the virion particle.