To determine if bile acids reflux into the esophagus in patients with gastroesophageal reflux disease and in normal subjects during physiological gastroesophageal reflux episodes, esophageal aspiration and pH monitoring were performed simultaneously in 16 patients with gastroesophageal reflux disease and 8 normal subjects. Esophageal samples were collected for 30 min in the fasting state and for 3 h (as hourly samples) after the ingestion of a test meal (egg salad sandwich, peaches, and milk). Bile acids were assayed by a standard enzymatic assay and also by a sensitive and specific assay, liquid secondary ion mass spectrometry. Bile acids were not detected by enzymatic assay in any of the fasting samples. In 6 of the 8 normal subjects and 15 of the 16 patients with gastroesophageal reflux disease, bile acid-like reactivity was detected by the enzymatic assay in postprandial samples. However, bile acid-like reactivity was also found by enzymatic assay in aliquots of the homogenized test meal in concentration similar to the highest concentrations detected in esophageal aspirates. Bile acids were not detected by the liquid secondary ion mass spectrometry method in any of the fasting or postprandial esophageal aspirates (limit of detection greater than 2 microM). However, this assay accurately identified samples to which exogenous bile acids were added as controls. Our results suggest that bile acids did not reflux into the esophagus of patients with gastroesophageal reflux disease whom we studied and caution must be exercised in the use of enzymatic assay for bile acids in postprandial gastrointestinal fluids, as the commonly used hydroxysteroid dehydrogenase assay is not specific for bile acids alone.