Serum antibodies reactive with streptococcal cell wall peptidoglycan (PG) and its peptide subunit (synthetic tetra-D-alanine) were measured by enzyme-linked immunosorbent assay (ELISA) in patients with rheumatoid arthritis (RA), juvenile rheumatoid arthritis (JRA), osteoarthritis and acute rheumatic fever (RF) compared with healthy subjects. Using 'checkerboard' titrations, anti-PG antibody in human serum was detected at a concentration of PG antigen at 10 micrograms per well with serum dilutions of 1:1,000. For measurement of anti-tetra-D-alanine antibody, the antigen, (D-Ala4)31 was used at 0.5 micrograms per well and sera were diluted to 1:200. When the IgG antibody levels to the PG and the tetra-D-alanine of the sera of patients with RA, JRA and RF were compared with sera from healthy subjects, the sera of the patients had significantly higher levels than did healthy subjects. Antibody that reacted with the PG in serum was absorbed with purified group-specific C-carbohydrate (A-CHO), but A-CHO was not capable of absorbing anti-(D-Ala4)31 antibodies. Therefore, the peptide subunit should be used as antigen in order to measure the specific antibody to PG. Both anti-PG and anti-tetra-D-alanine antibody in human sera primarily belonged to the IgG2 subclass.