Bacterial invasion of human mucosal cells is considered to be a primary event in the pathogenesis of a gonococcal infection. Here we report that cell surface heparan sulfate proteoglycans may play a role in the establishment of an infection, by functioning as receptors for the invasion-promoting gonococcal opacity protein adhesin. Chemical modification and enzymatic removal of proteoglycan receptors from cultured epithelial cells abolished opacity protein-associated gonococcal invasion, and mutant cell lines defective in proteoglycan synthesis were poor substrates for gonococcal attachment. The addition of purified receptor and receptor analogues totally blocked gonococcal entry into the cells. Heparin-affinity chromatography and receptor binding assays using recombinant bacteria producing defined opacity proteins and reconstituted receptor or purified receptor fragments as probes, identified one particular member of the opacity protein family (MS11-Opa30) as the primary ligand for this novel class of receptors for bacteria. Heparan sulfate proteoglycans with gonococcal binding activity were purified from various cell types derived from target tissues of gonococcal infection, including ME-180 endocervical cells and primary cultures of human corneal epithelium. The physico-chemical properties of the receptor indicate that it may belong to the syndecan proteoglycan family.