Airway pepsin has been increasingly used as a potentially sensitive and quantifiable biomarker for gastric-to-pulmonary aspiration, despite lack of validation in normal control subjects. This study attempts to define normal levels of airway pepsin in adults and distinguish between pepsin A (exclusive to stomach) and pepsin C (which can be expressed by pneumocytes). We performed a prospective study of 51 otherwise healthy adult patients undergoing elective extremity orthopedic surgery at a single tertiary-care academic medical center. Lower airway samples were obtained immediately following endotracheal intubation and just prior to extubation. Total pepsin and pepsin A concentrations were directly measured by an enzymatic activity assay, and pepsin C was subsequently derived. Pepsinogen/pepsin C was confirmed by Western blot analyses. Baseline characteristics were secondarily compared. In all, 11 (22%; 95% CI = 9.9%-33%) had detectable airway pepsin concentrations. All 11 positive specimens had pepsin C, without any detectable pepsin A. Pepsinogen/pepsin C was confirmed by Western blot analyses. In a multivariate logistic regression, men were more likely to have airway pepsin (OR, 12.71, P = .029). Enzymatically active pepsin C, but not the gastric-specific pepsin A, is frequently detected in the lower airways of patients who otherwise have no risk for aspiration. This suggests that nonspecific pepsin assays should be used and interpreted with caution as a biomarker of gastropulmonary aspiration, as pepsinogen C potentially expressed from pneumocytes may be detected in airway samples.