Previous characterization of Lyt mu 1/6, an endolysin from Streptomyces aureofaciens phage mu 1/6, suggested that the N-terminal domain is responsible for the catalytic activity of Lyt mu 1/6. Mutational analyses ( deletions and site-directed mutagenesis) demonstrated that lytic activity of Lyt mu 1/6 relies on the N-terminal part of about 200 amino acid residues. Various C-terminally truncated versions of Lyt mu 1/6 failed to cause lysis, indicating the necessity of the CBD for full enzyme activity. Functional analysis of the point mutants suggested that the residues K27, H31, E109, H176, and D184 were essential for lytic activity of the mu 1/6 endolysin. Further characterization of the purified Lyt mu 1/6 revealed that this endolysin is an N-acetylmuramoyl-L-alanine amidase which seems to be unrelated to any of the known conserved catalytic domains of phage endolysins or bacterial autolysins.