ABSTRACT We have studied the effect of mutations in the human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) sequence on tRNA 3 Lys genomic placement, i.e., the in vivo placement of primer tRNA 3 Lys on the HIV-1 primer binding site (PBS). HIV-1 produced from COS cells transfected with wild-type or mutant proviral DNA was used in this study. We have found that mutations in the amino acid sequences flanking the first Cys-His box in the NC sequence produce the maximum inhibition of genomic placement. A similar finding was obtained when the NC-facilitated annealing of primer tRNA 3 Lys to the HIV PBS in vitro was studied. However, since the genomic placement of tRNA 3 Lys occurs independently of precursor protein processing, the NC mutations studied here have probably exerted their effect through one or both of the precursor proteins, Pr55 gag and/or Pr160 gag-pol . One mutation in the linker region between the two Cys-His boxes, P31L, prevented packaging of both Pr160 gag-pol and tRNA 3 Lys and prevented the genomic placement of tRNA 3 Lys . Both packaging and genomic placement were rescued by cotransfection with a plasmid coding for wild-type Pr160 gag-pol . For other linker mutations [R7R10K11 S, R32G, and S3(32-34)], packaging of Pr160 gag-pol and tRNA 3 Lys was not affected, but genomic placement was, and placement could not be rescued by cotransfection with plasmids coding for either Pr55 gag or Pr160 gag-pol . After placement, the initiation of reverse transcription within extracellular virions is characterized by a 2-base DNA extension of the placed tRNA 3 Lys . This process requires precursor processing, and those NC mutations which showed the most inhibition of initiation were in either of the two NC Cys-His boxes. Destabilization of a U5 stem-A-rich loop immediately upstream of the PBS (through deletion of four consecutive A’s in the loop) did not affect the in vivo genomic placement of tRNA 3 Lys but resulted in the presence in the extracellular virus of longer cDNA extensions of tRNA 3 Lys , with a corresponding decrease in the presence of unextended and 2-base-extended tRNA 3 Lys .