Optical imaging based on intrinsic optical signals has been widely utilized in studies on the functional organization of various areas of the brain. Because of the small size of the optical signals and the large amount of background noise that is present, it is critical to be able to separate the stimulus-driven optical change from the background. In this study, we propose a novel method and apply it to the analysis of the orientation preference structure in cat area 17. The three criteria used to determine the activation included the amplitude of the optical change in the optical intensity, the statistical significance level of the change, and the temporal relationship between the optical change and the visual stimulation. In the activated region extracted by the new method, 82.4 ± 8.2% of the cells were electrophysiologically responsive to the stimuli that evoked the optical change. This was significantly higher than the 46.4 ± 7.1% value obtained when the activation region was extracted by the most widely used method, i.e. a differential map between two complementary stimuli, such as orthogonal orientations. Our newly proposed method provides a robust and an effective way to reduce manual operations used in the signal extraction process. Copyright © 2009 Institute of Electrical Engineers of Japan. Published by John Wiley & Sons, Inc.