Background: Staining dyes can provide a clear image in magnification colonoscopy, but not instantly. Indigocarmine (IC) shows contrast effect instantly, but this effect is not as reliable as the staining dyes. Acetic acid (AA) is cheap, easily available and safe. AA has been used in the evaluation of cervical and oesophageal mucosa, but its use has not been evaluated in magnification colonoscopy. The aim of this study was to evaluate the role of AA dye spray during magnification colonoscopy alone and in combination with IC dye. Methods: In one institution, 46 patients (F22, M24; age 56±17) entered into a prospective study of magnification colonoscopy, performed by a single endoscopist. The 46 consecutive patients were divided alternately into 2 groups; A (n=23) and B (n=23). In Group A, 1.5% AA was initially sprayed on to all detected lesions, followed by spray with 0.2% IC. In Group B, the order was reversed, with IC preceding AA. Pit pattern image was assessed in real time after the spraying of each consecutive dye. Pit patterns were evaluated based upon Kudo's classification. The time required to obtain a clear image after the first dye spray was measured. The effect of the second dye was evaluated based upon whether the pit pattern image following additional dye spray was clearer than that after the first dye alone. Results: 37 adenomas (AD), 36 hyperplastic polyps (HP), 5 normal mucosa, 4 inflammatory polyps and 1 serrated adenoma were detected. The 73 lesions comprised of AD and HP alone were subjected to further analysis. In group A, 20 AD and 23 HP were detected, and accuracies after AA spray were 95% with AD and 96% with HP. After subsequent IC spray, 37% showed enhanced images, and accuracies increased to 100% in AD and 97% in HP. In group B, 17 AD and 13 HP were detected, and accuracies after IC spray were 76% with AD and 92% with HP. After subsequent AA spray, images were enhanced in 70% with increases in accuracy to 94% in AD and 100% in HP. In both groups, the mean time required to obtain an initial clear image was 14 seconds. Conclusion: The use of AA spray instantly improves pit pattern image during magnification chromo-colonoscopy and improves the accuracy of histological prediction of colorectal polyps. This method could be easily applied to routine magnification colonoscopy.