Bile reflux may play a key role for esophageal carcinogenesis in reflux disease. In search for bile-specific effects, the animal model of esophageal cancer was applied in a mutagenesis assay. Big Blue transgenic mice were operated with microsurgical techniques. Seven had total gastrectomy with esophagojejunostomy creating esophageal reflux of bile and five had a sham operation. After 24 weeks, the mutation frequency (MF) was measured through standard Big Blue mutagenesis assay in the esophageal mucosa and the duodenum as control. Esophageal reflux resulted in esophagitis in the distal esophagus. The MF in esophageal mucosa was 1.6 times higher in animals with reflux than in sham-operated animals; it was identical in the duodenum. In conclusion, the mutagenic potential of bile reflux has been confirmed. However, mechanisms of carcinogenesis in the esophageal cancer model other than chronic inflammation could not be identified because of the only moderately increased MF.