• Background Although assessment for aspiration of small volumes of gastric contents in tube-fed patients receiving mechanical ventilation is important, available methods for this purpose are not wholly satisfactory. A potential method is immunoassay of tracheal secretions for the gastric enzyme pepsin. • Objectives To determine the frequency with which pepsin in suctioned tracheal secretions from acutely ill, tube-fed patients receiving mechanical ventilation could be detected via an immunoassay. • Methods A convenience sample of 136 specimens of suctioned tracheal secretions was collected from 30 acutely ill, tube-fed adults receiving mechanical ventilation. Multiple samples were obtained from 26 of the 30 patients (range, 2–11 per subject). An immunoassay with rooster polyclonal antibodies to purified human pepsin was used to detect pepsin in the secretions. • Results Fourteen specimens tested positive for pepsin. Secretions from 5 patients accounted for the 14 pepsin-positive results. A significant relationship was found between the position of the head of the bed and the presence of pepsin in tracheal secretions (P< .001). Of the 14 pepsin-positive specimens, 13 (92.9%) were obtained from subjects in a flat position. • Conclusions A pepsin immunoassay can be used to detect pepsin in human tracheal secretions. If pepsin in tracheal secretions is considered an indicator of aspiration of gastric contents, aspiration occurred in 5 of the 30 subjects. A flat position is strongly associated with the presence of pepsin in tracheal secretions.