Reflux esophagitis may result from the action of both acid and non-acid agents. The aim of this study was to test a new system able to measure the quantity of the bilirubin contained in the esophageal lumen. The analysis of esophageal reflux composition was conducted in two phases. In the first bile and pancreatic enzyme, concentration of 136 fluid samples obtained with ambulatory esophageal long-term reflux aspiration test were measured. For the second, the total bilirubin content of each sample was measured in vitro with a fiberoptic probe (Bilitec 2000, Synetics Medical Inc., Sweden). Studies were performed on 48 subjects: 43 patients with esophageal reflux and five healthy volunteers. The results of both techniques were then compared. Higher concentration of bile and pancreatic enzymes were found in esophageal fluid samples of patients with endoscopic esophagitis. Bile and pancreatic enzyme concentrations of esophageal fluid samples were higher in patients after gastrectomy compared to patients with intact stomachs. There was a significant correlation between the total bilirubin concentration of fluid specimens and the fiberoptic probe reading of bilirubin (r = 0.72, P < 0.001). The presence of bilirubin and bile acids within the esophageal refluxate can be determined reliably with continuous fiberoptic measurement. The correlation between total bilirubin content and the concentrations of pancreatic enzymes contained in the esophageal refluxate suggests that bilirubin is a good tracer for non-acid, duodenal or intestinal reflux in the esophagus.