To study the initiation of human immunodeficiency virus type 1 reverse transcription, we have used the viral nucleocapsid protein (NC7) to anneal tRNA3Lys primer onto viral genomic RNA and have then eliminated NC7 from this primer-template complex by digestion with proteinase K and phenol-chloroform extraction of residual protein. Our data show that saturating concentrations of NC7 resulted in the formation of an active tRNA-template complex that yielded enhanced production of full-length negative-strand strong-stop DNA [(-)ssDNA] and that this complex remained active even after the elimination of NC7. While both of the two Zn finger motifs found within NC7 were essential for efficient elongation, NC protein that contained a point mutation in the first Zn finger or that was devoid of both Zn fingers yielded primer-template complexes that could still be initiated in 1-base-extension assays. In contrast, the use of heat annealing to produce primer-template complexes resulted in proportions of full-length (-)ssDNA lower than those seen with NC protein, and the addition of NC protein to such preformed primer-template complexes was able to reverse this defect only to a marginal extent.