ABSTRACT Staphylococcus aureus and coagulase-negative staphylococci are microorganisms most frequently isolated from orthopedic-implant-associated infections. Their capacity to maintain these infections is thought to be related to their ability to form adherent biofilms. Poly- N -acetyl-β-(1,6)-glucosamine (PNAG) is an important constituent of the extracellular biofilm matrix of staphylococci. In the present study, we explored the possibility of using PNAG as an antigen for detecting antibodies in the blood sera of patients with staphylococcal orthopedic-prosthesis-associated infections. First, we tested the presence of anti-PNAG antibodies in an animal model, in the blood sera of guinea pigs that developed an implant-associated infection caused by biofilm-forming, PNAG-producing strains of Staphylococcus epidermidis . Animals infected with S. epidermidis RP62A showed levels of anti-PNAG immunoglobulin G (IgG) significantly higher than those of the control group. The comparative study of healthy individuals and patients with staphylococcal prosthesis-related infections showed that (i) relatively high levels of anti-PNAG IgG were present in the blood sera of the healthy control group, (ii) the corresponding levels in the infected patients were slightly but not significantly higher, and (iii) only 1 of 10 patients had a level of anti-PNAG IgM significantly higher than that of the control group. In conclusion, the encouraging results obtained in the animal study could not be readily applied for the diagnosis of staphylococcal orthopedic-prosthesis-related infections in humans, and PNAG does not seem to be an appropriate antigen for this purpose. Further studies are necessary to determine whether the developed enzyme-linked immunosorbent assay method could serve as a complementary test in the individual follow-up treatment of such infections caused by PNAG-producing staphylococci.