Intracellular killing of Staphylococcus aureus by alveolar macrophages is known to be enhanced by exposure to alveolar lining material. Because this material may have a role in pulmonary host defenses, we have studied its effect on pneumococci and other nonstaphylococcal organisms. Alveolar lining material from rats caused rapid killing and lysis of pneumococci. The antipneumococcal activity was localized to the surfactant-containing fraction of the fluid and was not affected by trypsin. Phospholipid extracts of the surfactant fraction or purified lamellar bodies killed pneumococci. Lysis of pneumococci by the surfactant fraction appeared to be mediated by a detergent-like activation of pneumococcal autolysin, in that bacteriolysis was prevented by substitution of ethanolamine for choline in pneumococcal cell walls, and a pneumococcal transformant that lacked autolysin was not lysed. The surfactant fraction readily killed pneumococci containing ethanolamine or the autolysin-defective transformant, and studies with tritiated methyl-D-glucose loading and release showed that killing was associated with increased bacterial cell membrane permeability. Bactericidal activity (without lysis) was observed with several nonpneumococcal gram-positive bacteria, including Streptococcus viridans, unspeciated respiratory streptococci, Streptococcus pyogenes, Streptococcus bovis, and Bacillus species. Purified diacylphospholipids had no antibacterial activity, however, a lysophospholipid, palmitoyl lysophosphatidylcholine, had many properties resembling the surfactant-containing fraction of lavage, including autolysin-mediated pneumococcal lysis, altered cell membrane permeability, and antibacterial activity against several gram-positive bacteria.