The dissemination of tracheal tube biofilm into the mechanically ventilated lung has been proposed as a contributory factor in the pathogenesis of ventilator-associated pneumonia. In the present study, conventional light microscopy, confocal laser scanning microscopy, and scanning electron microscopy were used to examine luminal tracheal tube biofilm in tubes from ten consecutive medical intensive care patients. Biofilms also were cultured. No tube contained a predominantly microbial aggregate. Microorganisms were either dispersed throughout the biofilm or restricted to the most superficial layer. Neutrophil polymorphonuclear cells were present in all biofilms in a pattern suggesting that a layering or stratification had taken place. The distribution of neutrophils and microorganisms was consistent with a progressive accretion of respiratory secretions, rather than formation of a predominantly microbial biofilm.