The pathophysiologic effect of duodenal contents in the refluxed gastric juice of patients with gastroesophageal reflux disease (GERD) is controversial. We evaluated the composition of the refluxed gastric juice in 43 normal volunteers and 52 patients with GERD using a newly developed device that allows ambulatory esophageal aspiration. The findings were correlated with the results of 24-hour esophageal pH monitoring and the presence of complications of GERD. Compared with bile concentrations in normal volunteers, the total bile acid concentration in the reflux aspirates was higher in patients with GERD (p < 0.01). There was a significant correlation between the bile acid concentration in the aspirates and the percentage of time the pH was above 7 on ambulatory 24-hour esophageal pH monitoring (r = 0.59, p = 0.006), and both were highest during the night (p < 0.01). The bile acid concentration and the percentage of time pH was greater than 7 were particularly increased in patients with strictures or Barrett's esophagus (p < 0.01). Both an increased bile acid concentration in aspirates and the percentage of time with pH greater than 7 on pH monitoring were observed primarily in patients with a destroyed gastroduodenal barrier [status post Billroth II resection (BII), Billroth I resection (BI), or pyloroplasty] or after cholecystectomy. An increased bile acid concentration also occurred in a substantial number of patients without previous foregut surgery, although this did not usually result in an increase in the time that pH was above 7. These data suggest that contamination of the refluxed gastric juice with bile acids predisposes the patient to the development of strictures and Barrett's esophagus. An increased time that pH is greater than 7 on esophageal pH monitoring indicates biliary reflux and occurs primarily after previous foregut surgery. A normal-time pH above 7 does, however, not exclude contamination of the refluxed gastric juice with duodenal contents.