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Aspiration of gastric contents is a relatively common cause of acute and chronic pulmonary disease. However, a reliable method of diagnosing recurrent aspiration is currently lacking. The aim of this study was to determine whether the presence of gastric pepsin in tracheal aspirates of infants and children might be used as a reliable marker of the microaspiration of refluxed gastric contents.Ninety-eight children undergoing general anesthesia and tracheal intubation participated in the study. Sixty-four of 98 children underwent endoscopy for clinically significant gastroesophageal reflux. Thirty-four children from routine operative lists were nonreflux controls. These two groups were further subdivided based on the presence or absence of associated respiratory symptoms. After endotracheal intubation, tracheal aspirates were obtained and subsequently assayed for gastric pepsin using a fluoroscein isothiocyanate casein.Pepsin was detected in 7 of 27 children with reflux symptoms alone and in 7 of 8 of those with chronic respiratory symptoms. In addition, pepsin was present in 31 of 37 children with a history of both reflux and chronic respiratory symptoms. Tracheal pepsin was not detected in any of the 26 children without gastroesophageal reflux or respiratory symptoms. Tracheal pepsin was found significantly more frequently in children with reflux symptoms than in those without, particularly in children with both reflux and respiratory problems.Tracheal pepsin assay as a reliable marker of gastroesophageal reflux aspiration.