Urokinase-type plasminogen activator (uPA), a proteinase which activates plasminogen by cleaving at -CPGR(arrow downward)V-, was shown to cleave the V3 loop in recombinant gp120 of human immunodeficiency virus type 1 (HIV-1) IIIB and MN strains, as well as a synthetic, cyclized peptide representing the clade B consensus sequence of V3. Proteolysis occurred at the homologous -GPGR(arrow downward)A-, an important neutralizing determinant of HIV-1. It required soluble CD4 and was prevented by inhibitors of uPA but not by inhibitors of likely contaminating plasma proteinases. It was accelerated by heparin, a known cofactor for plasminogen activation. In immune capture experiments, tight binding of uPA to viral particles, which did not depend on CD4, was also demonstrated. Active site-directed inhibitors or uPA diminished this binding, as did a neutralizing antibody to V3. Addition of exogenous uPA to the laboratory-adapted IIIB strain of HIV-1, the macrophage-tropic field strains JR-CSF and SF-162, or a fresh patient isolate of indeterminate tropism, followed by infection of macrophages with the various treated viruses, resulted in severalfold increases in subsequent viral replication, as judged by yields of reverse transcriptase activity and p24 antigen, as well as incorporation, as judged by PCR in situ. These responses were reversible by inhibitors or antibodies targeting the proteinase active site or the V3 loop. We propose that uPA, a transcriptionally regulated proteinase which is upregulated when macrophages are HIV infected, can be bound and utilized by the virus to aid in fusion and may be an endogenous component that is critical to the infection of macrophages by HIV-1.