Cigarette smoking is a major risk factor for diseases including cardiovascular disease, lung cancer, and COPD. One of the key functions of lung epithelial cells is to serve as a physical barrier against insults including cigarette smoke. Chronic smoking induces a state of chronic inflammation and oxidative stress, resulting in epithelial barrier disruption, which leads to increased lung permeability and culminates in tissue damage and remodeling, contributing to smoking-induced lung diseases. We evaluated lung barrier function in a single-center, ambulatory, clinical study. Lung permeability, measured as the half-life (T1/2) of inhaled 99mTC-DTPA, was assessed in seventeen subjects consisting of six Smokers (SMK), five Moist Snuff Consumers (MSC), and six Non-Tobacco Consumers (NTC). Half time clearance of 99mTC-DTPA from the lungs was measured at baseline, and approximately 7 and 14 days later. Right lung images were captured with a gamma-camera after inhalation of 99mTC-DTPA. Smokers, relative to MSC and NTC, exhibited significantly faster clearance of the inhaled probe (shorter T1/2), indicating increased lung permeability. NTC and MSC had similar clearance times of the probe (longer T1/2), suggesting that moist snuff use does not perturb lung permeability. The altered lung barrier function in smokers is a contributing factor to smoking-related diseases such as lung cancer and COPD. Altered lung barrier function may serve as a biomarker of potential harm related to tobacco use.