Previously we demonstrated that murine retroviral Gag proteins associate with a cellular motor protein, KIF-4. Using the yeast two-hybrid assay, we also found an association of KIF-4 with Gag proteins of Mason-Pfizer monkey virus (MPMV), simian immunodeficiency virus (SIV), and human immunodeficiency virus type 1 (HIV-1). Studies performed with mammalian cell systems confirmed that the HIV-1 Gag protein associates with KIF-4. Soluble cytoplasmic proteins from cells infected with recombinant vaccinia virus expressing the entire Gag-Pol precursor protein of HIV-1 or transfected with HIV-1 molecular clone pNL4-3 were fractionated by sucrose gradient centrifugation and further separated by size-exclusion and anion-exchange chromatographies. KIF-4 and HIV-1 Gag cofractionated in both chromatographic separations. Immunoprecipitation assays have also verified the KIF-4–Gag association. KIF-4 binds mainly to the Gag precursor (Pr55 Gag) and a matrix-capsid processing intermediate (Pr42) but not to other processed Gag products. The binding of Gag is mediated by a domain of KIF-4 proximal to the C terminus. These results, and our previous studies, raise the possibility that KIF-4 may play an important role in retrovirus Gag protein transport.