To investigate the possible antibacterial properties of human alveolar lining material (ALM), we obtained ALM and pulmonary alveolar macrophages (PAM) by bronchoalveolar lavage of healthy nonsmokers. Alveolar lining material was isolated by centrifugation or micropore filtration; electron microscopy revealed lamellar bodies, and lipid analysis showed that 98% of the lipid fraction was phospholipid. No free fatty acids were detected. Streptococcus pneumoniae and non-typable Haemophilus influenzae (NTHI) died spontaneously in PBS at a mean rate of log₁₀ 0.75 and 0.95 in 90 min, respectively; the addition of ALM appeared to exert a slight protective effect, and at higher concentrations supported replication of NTHI. There was no difference in the uptake of the bacteria by PAM when ALM was present. Phagocytosed NTHI were killed rapidly and completely within 60 min by PAM with or without ALM. A greater proportion of S. aureus were killed by PAM alone than in the presence of ALM. Alveolar lining material from healthy humans thus appears to have no demonstrable effect on host defense against these bacteria. The differences between our results and those of earlier studies using ALM from rats may relate to interspecies differences in the composition of ALM.