The tRNAs used to prime reverse transcription in human immunodeficiency virus type 1 (HIV-1), Rous sarcoma virus (RSV), and Moloney murine leukemia virus (Mo-MuLV) are, tRNA(Trp), and tRNA(Pro), respectively. Using antibodies to the three cognate human aminoacyl-tRNA synthetases, we found that only lysyl-tRNA synthetase (LysRS) is present in HIV-1, only tryptophanyl-tRNA synthetase (TrpRS) is present in RSV, and neither these two synthetases nor prolyl-tRNA synthetase (ProRS) is present in Mo-MuLV. LysRS and TrpRS are present in HIV-1 and RSV at approximately 25 and 12 molecules/virion, respectively. These results support the hypothesis that, in HIV-1 and RSV, the cognate aminoacyl-tRNA synthetase may be used as the signal for targeting the selective packaging of primer tRNAs into retroviruses. The absence of ProRS in Mo-MuLV is consistent with reports that selective packaging of tRNA(Pro) in this virus is less important for achieving optimum annealing of the primer to Mo-MuLV genomic RNA.