By itself, lipoteichoic acid (LTA) obtained from S. pyogenes, S. aureus, or E. hirae poorly stimulated cytokine production by macrophages, whereas in the presence of anti-polyglycerol phosphate (PGP), the cells secreted significant amounts of IL-6. Two peptides constructed from the deduced sequence of the selected anti-PGP phage-antibody's complementary-determining region 3 of the variable heavy chain (V(H)-CDR3) reacted specifically with PGP. The monomeric form of the peptides markedly inhibited cytokine production by macrophages pretreated with LTA and anti-LTA. In contrast, the polyvalent form of biotinylated peptides complex with streptavidin-induced cytokine production by the LTA-treated macrophages. The data taken together support the concept that cross-linking of macrophage-bound LTA by anti-PGP is required for cytokine release by these cells. Importantly, these studies identified small, PGP-reactive peptides as potential tools in reducing this proinflammatory process.