The epithelium lining the conducting airways restricts the movement of inhaled foreign materials into the interstitial and circulatory space. In the present study, we have investigated the effects of graded doses of inhaled whole cigarette smoke on this function by measuring the rate of movement of horseradish peroxidase (HRP) from the airways lumen across the tracheal epithelium into blood, by assessing the penetration of HRP into tracheal epithelium using transmission electron microscopy, and by examining the morphology of tracheal epithelial tight junctions using the freeze-fracture technique. These studies showed that 5, 20, and 100 puffs of whole smoke increased penetration of HRP from the tracheal lumen into blood in a dose-dependent fashion. Transmission electron microscopy showed that HRP penetrated the tracheal epithelium only in the 100-puff exposure group while the freeze-fracture studies showed progressive disruption of epithelial tight junction beginning with a 100-puff exposure and becoming more extensive after the 200-puff exposure. We conclude that inhaled cigarette smoke damages the mucosal barrier and causes increased permeability to HRP by disrupting the intercellular tight junctions and that measuring the appearance of HRP in blood is more sensitive than electron microscopy in assessing this damage.