To study in vivo tRNA(3)(Lys) genomic placement and the initiation step of reverse transcription in human immunodeficiency virus type 1, total viral RNA isolated from either wild-type or protease-negative (PR(-)) virus was used as the source of primer tRNA(3)(Lys)/genomic RNA templates in an in vitro reverse transcription assay. At low dCTP concentrations, both the rate and extent of the first nucleotide incorporated into tRNA(3)(Lys), dCTP, were lower with PR(-) than with wild-type total viral RNA. Transient in vitro exposure of either type of primer/template RNA to NCp7 increased PR(-) dCTP incorporation to wild-type levels but did not change the level of wild-type dCTP incorporation. Exposure of either primer/template to Pr55(gag) had no effect on initiation. These results indicate that while Pr55(gag) is sufficient for tRNA(3)(Lys) placement onto the genome, exposure of this complex to mature NCp7 is required for optimum tRNA(3)(Lys) placement and initiation of reverse transcription.