Sera from patients with verified systemic staphylococcal infection contained antibodies reactive with peptidoglycan (PG) from Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Micrococcus lysodeikticus, Bacillus subtilis, and Escherichia coli. The presence of anti-PG cross-reactive antibodies was verified in patient sera by inhibition studies with the various bacterial PGs. Antibodies to nonstaphylococcal PGs were also elevated in sera from rabbits immunized with S. aureus PG. Antibodies to S. aureus PG were removed with the synthetic peptide analogs of S. aureus PG, the L-Lys-D-Ala-D-Ala, L-Lys-D-Ala, and (Gly)5 determinants, as well as with an S. aureus PG peptide fragment containing the determinants D-Ala-D-Ala and L-Lys-D-Ala. Isolated antibodies to the PG peptides, both synthetic and native, were reactive with S. aureus and S. epidermidis PGs. The antibodies to the D-Ala-D-Ala and the L-Lys-D-Ala determinants were also reactive with S. pyogenes PG, but not with PGs from M. lysodeikticus, B. subtilis, and E. coli.