Lysozyme is abundant in respiratory secretions and may play a role in lung host defenses. Mechanisms by which lysozyme killed Streptococcus pneumoniae, an important respiratory pathogen, were studied. Lysozyme caused optical clearing of pneumococcal suspensions and released fragments containing [3H]choline from their cell walls. Electron micrographs revealed wide-spread cell wall destruction and bacteriolysis. Breakdown of the cell wall appeared to be mediated mostly by the major pneumococcal autolysin, N-acetylmuramoyl-L-alanine amidase, because it was blocked by phosphorylcholine, a specific inhibitor of amidase, or by substitution of ethanolamine for choline in the cell wall. Blockade of amidase did not greatly increase survival of lysozyme-treated pneumococci on blood agar. Pneumococci in which amidase was blocked appeared intact immediately after treatment with lysozyme, but when they were reincubated at 37 degrees C in fresh culture medium they swelled and lysed. Thus, widespread triggering of the major pneumococcal autolysin is not essential for the bactericidal effect of lysozyme.