Introduction The aim of the study was to analyse pH-and bile-monitoring data in patients with Barrett's esophagus and in age-and gender-matched controls. Subjects and Methods Twenty-four consecutive Barrett's patients (8 females, 16 males, mean age 57 years), 21 patients with esophagitis (10 females, 11 males, mean age 58 years), and 19 healthy controls (8 females, 11 males, mean age 51 years), were included. Only patients underwent endoscopy with biopsy. All groups were investigated with manometry, gastric and esophageal 24-h pH, and simultaneous bile monitoring according to a standardized protocol. A bilirubin absorption >0.25 was determined as noxious bile reflux. The receiver operator characteristic (ROC) method was applied to determine the optimal cutoff value of pathologic bilirubin levels. Results Of Barrett's patients, 79% had pathologic acidic gastric reflux (pH<4 >5% of total measuring time). However, 32% of healthy controls also had acid reflux (p<0.05) without any symptoms. The median of esophageal bile reflux was 7.8% (lower quartile (LQ)-upper quartile (UQ)=1.6-17.8%) in Barrett's patients, in patients with esophagitis, 3.5% (LQ-UQ= 0.1-13.5), and in contrast to 0% (LQ-UQ=0-1.0%) in controls, p=0.001. ROC analysis showed the optimal dividing value for patients at more than 1% bile reflux over 24 h (75% sensitivity, 84% specificity). Conclusion An optimal threshold to differentiate between normal and pathological bile reflux into the esophagus is 1% (24-h bile monitoring with an absorbance >0.25).