The biological activities of the cell walls of bacteria having different types of peptidoglycans, and those of stereoisomers and analogs of muramyl dipeptide (MDP), of N-acetylglucosaminyl-beta(1-4)-N-acetylmuramyl tetrapeptides having different L- and D-amino acids at the COOH-terminus, and of 6-O-acyl-MDPs were examined to elucidate the relationship between structure and activity. Replacement of the L-alanine residue of MDP with glycine and replacement of the D-isoglutamine residue with L-isoglutamine, L-glutamic acid, and D-isoasparagine, but not with D-glutamic acid, caused a marked decrease in the biological activities of the MDP molecule. Test disaccharide tetrapeptides, irrespective of the configuration of COOH-terminal amino acid, showed strong immunoadjuvant activity and stimulation of macrophages, whereas those having COOH-terminal L-amino acids exhibited greater pyrogenicity, induction of acute joint inflammation, and hemorrhagic necrosis at a primed site than those having COOH-terminal D-amino acids. Introduction of an alpha-branched higher fatty acid to the muramic acid residue resulted in the disappearance of pyrogenicity after i.v. injection, an increase of adjuvanticity, and a loss of dependence on administration vehicles. The lack of the immunopotentiating activity (adjuvanticity) in cell walls from group B-type bacterial species was explained by the combined inhibitory effects of the replacement of the L-alanine residue by glycine and involvement of the alpha-carboxyl group of the D-glutamic acid residue in linking with neighboring peptide subunits.