The serine esterase TL2 from human T4+ lymphocytes is a binding component to HIV-1 glycoprotein gp120 and seems to play a role in the HIV-1 infection mechanism. Recombinant variants of the Kunitz-type serine proteinase inhibitor aprotinin were investigated for their ability to inhibit tryptase TL2 and the binding of gp120 to this enzyme. Furthermore, the viral replication of HIV-1 was investigated H9 cell cultures under the influence of recombinant aprotinin and bikunin variants. In contrast to native aprotinin, the recombinant variant [Arg15, Phe17, Glu52] aprotinin with a reactive-site sequence homologous to the V3 loop of HIV-1 gp120 showed a specific inhibition of tryptase TL2 (> 80%). However, the [Leu15, Phe17, Glu52] aprotinin variant with hydrophobic subsites was the most potent inhibitor of the binding of gp120 to tryptase TL2 (68%). Our results show that the enzyme activity of purified tryptase TL2 is inhibited not only by variants with basic amino acids, but also those with hydrophobic residues in the reactive-site region. Therefore, tryptase TL2 is not a typical trypsin-like or chymotrypsin-like protease. Investigations on inhibition of HIV-1 replication in H9 cell cultures showed that tryptase TL2 is involved in the mechanism of virus internalization into human lymphocytes. The [Leu15, Phe17, Glu52] aprotinin showed a significant retardation of syncytium formation over a period of 5 days in a 1 micro M concentration. Similar investigations were performed with recombinant variants of bikunin, the light chain of human inter-alpha-trypsin inhibitor. Only the single-headed variant [Arg94] delta 2 bikunin inhibited slightly the syncytium formation over a period of 2 days in a 2.2 micro M concentration. Wild-type bikunin and all full-length variants showed no effect, possibly due to steric hindrance by the second domain of the double-headed inhibitor.