During human immunodeficiency virus type 1 (HIV-1) assembly, tRNA(Lys) isoacceptors are selectively incorporated into virions and tRNA(Lys)3 is used as the primer for reverse transcription. We show herein that the tRNA(Lys)-binding protein, lysyl-tRNA synthetase (LysRS), is also selectively packaged into HIV-1. The viral precursor protein Pr55gag alone will package LysRS into Pr55gag particles, independently of tRNA(Lys). With the additional presence of the viral precursor protein Pr160gag-pol, tRNA(Lys) and LysRS are both packaged into the particle. While the predominant cytoplasmic LysRS has an apparent M(r) of 70,000, viral LysRS associated with tRNA(Lys) packaging is shorter, with an apparent M(r) of 63,000. The truncation occurs independently of viral protease and might be required to facilitate interactions involved in the selective packaging and genomic placement of primer tRNA.