Site-directed mutagenesis experiments designed to identify the active site of Bacillus licheniformis endo-β-1,3-1,4-d-glucan 4-glucanohydrolase (β-glucanase) have been performed. Putative catalytic residues were chosen on the basis of sequence similarity analysis to viral and eukaryotic lysozymes. Four mutant enzymes were expressed and purified from recombinan: E. coli and their kinetics analysed with barley β-glucan. Replacement of Glu¹³⁴ by Gin produced a mutant (E134Q) that retains less than 0.3% of the wild-type activity. The other mutants, D133N, E160Q and D179N, are active but show different kinetic parameters relative to wild-type indicative of their participation in substrate binding and transition-state complex stabilization. Glu¹³⁴ is essential for activity; it is comprised in a region of high sequence similarity to the active site of T4 lysozyme and matches the position of the general acid catalyst. These results strongly support a lysozyme-like mechanism for this family of Bacillus β-glucan hydrolases with Glu¹³⁴ being the essential acid catalyst.