Gp181 (2237 amino acids) of Pseudomonas aeruginosa bacteriophage phiKZ (Myoviridae) is a structural virion protein, which bears a peptidoglycan hydrolase domain near its C-terminus. This protein is supposed to degrade the peptidoglycan locally during the infection process. Nine deletional mutants allowed delineation of the peptidoglycan hydrolase domain between amino acids 1880-2042 (gp181M8) and analysis of its biochemical properties. Gp181M8 tolerates a high ionic strength (>320mM) and is less sensitive to long thermal treatments compared to the similar phiKZ endolysin. Gp181M8 lysed all tested outer membrane-permeabilized Gram-negative species. The C-terminal distal end (amino acids 2043-2237) enhances the specific activity of gp181M8 threefold, resulting in a twelve times higher activity than commercial hen egg white lysozyme. These biochemical properties suggest that this novel peptidoglycan hydrolase domain may be suitable for enzybiotic applications.