It has been suggested that the third variable domain (V3) loop of human immunodeficiency virus type 1 (HIV-1) gp120 has to interact with a cell-surface-associated protease(s) that acts as a cofactor after binding of gp120 to the CD4 receptor during entry of HIV-1 into susceptible cells. We isolated the membrane-associated serine protease, tryptase TL2, from human CD4-positive lymphocytes. This enzyme specifically binds gp120 through interaction with its V3 domain. To investigate the role of tryptase TL2 in HIV infection, we examined the affinity of the interaction and the proteolytic susceptibility of various recombinant gp120 expressed in mammalian cells to the enzyme, and we determined the cleavage sites. Tryptase TL2 bound gp120 with an apparent dissociation constant of 38 nM. The affinity was lower than that of gp120 for CD4 which suggests that gp120 initially binds to CD4, followed by interaction with tryptase TL2 which is localized close to CD4 on the cell surface. After binding, tryptase TL2 cleaved recombinant gp120 expressed in mammalian cells into two protein species of 70 kDa and 50 kDa but did not cleave gp120 expressed in insect cells, which indicates that the structure of the oligosaccharides linked to the polypeptide backbone of gp120 affects the proteolytic susceptibility. Cleavage was specifically inhibited by a neutralizing antibody against the V3 loop. Cleavage-site determination revealed that tryptase TL2 cleaved gp120 at various sites in the V3 in a strain-dependent manner. The amino acid variability at the cleavage site(s) in almost all HIV-1 isolates was restricted to amino acids which are susceptible to the chymotryptic and/or tryptic activities of tryptase TL2.