Background: Image-enhanced endoscopy has been increasingly useful for esophageal cancer screening. Although iodine staining is currently the standard modality for the early diagnosis of esophageal cancer, conventionally used concentrations often induce mucosal irritation, leading to discomfort. The aim of the study was to determine the optimal FICE setting and iodine concentration required for maximum color enhancement for the endoscopic diagnosis of superficial esophageal cancer. Methods: Four esophageal specimens with squamous cell carcinoma that were surgically resected were investigated. The color difference between iodine-stained and background mucosa was evaluated using the 10 preset flexible spectral imaging color enhancement (FICE) wavelength combinations. In addition, the optimum wavelength that best reflected the widest color difference was calculated with and without FICE, and the color differences elicited by different iodine concentrations (from 0.1% to 0.8%) were evaluated with FICE and compared to 1% iodine without FICE (the control). Results: Maximum color differences were observed using the FICE7 wavelength values. Color enhancement with FICE0, 1, 5 and 7 using a concentration of 0.2% iodine solution or greater was significantly higher than that with the standard 1% iodine concentration (p &lt
0.05). Conclusions: Low-concentration iodine staining with image-enhanced endoscopy is a useful diagnostic modality that can reduce the degree of discomfort experienced by patients.