The matrix (MA) protein of human immunodeficiency virus type 1 (HIV-1) plays a critical role in virion morphogenesis and fulfills important functions during the early steps of infection. In an effort to identify cellular partners of MA, a Saccharomyces cerevisiae two-hybrid screen was utilized. A specific interaction between MA and HO3, a putative histidyl-tRNA synthetase, was demonstrated in this system. HO3-specific mRNA was detected in several tissues relevant for HIV infection, such as spleen, thymus, and peripheral blood lymphocytes, as well as in a number of T-lymphoid-cell lines. The binding of MA to HO3 was confirmed in transfected cells by coimmunoprecipitation. This interaction was abrogated by replacing two lysine residues at positions 26 and 27 of MA by threonine (MA(KK27TT)). HO3 localized both to the cytoplasm and to the nucleus of acutely transfected 293T cells. When overexpressed in HIV-1-producing cells, HO3 was incorporated into wild-type virions but not in ones containing the dilysine-mutated variant of MA. Correspondingly, overexpression of HO3 in virus producer cells enhanced the infectivity of wild-type but not MA(KK27AA) HIV-1 particles. The stimulating effect of HO3 was independent from the presence of Envelope, Vpr, or Vpu. Taken together, these results suggest that HO3, through its recognition of MA, plays a role in the life cycle of HIV-1.