Abstract Several phage-encoded peptidoglycan hydrolases have been found to share a conserved amidase domain with a variety of bacterial autolysins ( N-acetylmuramoyl- l-alanine amidases), bacterial and eukaryotic glutathionylspermidine amidases, γ- d-glutamyl- l-diamino acid endopeptidase and NLP/P60 family proteins. All these proteins contain conserved cysteine and histidine residues and hydrolyze γ-glutamyl-containing substrates. These cysteine residues have been shown to be essential for activity of several of these amidases and their thiol groups apparently function as the nucleophiles in the catalytic mechanisms of all enzymes containing this domain. The CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) superfamily includes a variety of previously uncharacterized proteins, including the tail assembly protein K of phage λ. Some members of this superfamily are important surface antigens in pathogenic bacteria and might represent drug and/or vaccine targets.