We used optical imaging of intrinsic signals to characterize the functional representations of mystacial vibrissae (whiskers) in rat somatosensory cortex. Stimulation of individual whiskers for 2 s at 5 Hz resulted in a discrete area of functional activity in the cortex. Images of whisker representations were collected both through the dura and through a thinned skull. We characterized the functional representation of a whisker both spatially and temporally with twodimensional images and three-dimensional surface plots of intrinsic signal development in the cortex in response to whisker stimulation. Single unit recordings verified that the representation of the whisker obtained with optical imaging corresponded with the electrophysiological response area of that whisker in the cortex. Lesions in the center of the functional activity were found to be in the center of the dense cytochrome oxidase patch for the corresponding whisker. In addition, a 3 x 3 matrix of whiskers was stimulated and the distances between the centers of the imaged representations and the distances between the centers of the layer IV cytochrome oxidase staining of the nine whiskers were found to be highly correlated (r = 0.98). This study shows a striking correspondence among imaging, physiology, and anatomy in the rat somatosensory cortex. Furthermore, the ability to use optical imaging through a thinned skull should allow investigations into the long-term changes in a sensory representation within a single animal.In vivo characterization of the functional organization of a sensory system, combined with knowledge about its underlying anatomical structure, should facilitate the elucidation of structure-function relationships. This study investigates the relationship between structure and function in the adult rat somatosensory cortex and uses optical imaging of intrinsic signals to establish the normal functional organization. Our long-term goals are to investigate changes in the functional organization of the cortex (i.e., plasticity) before, during, and after manipulations to the system. The ability to use intrinsic signal imaging to investigate cortical plasticity has two requirements. (i) Optical imaging should reliably describe the normal functional organization of the cortex and its relationship to structure.(ii) The imaging should be noninvasive to the cortex to allow repeated sampling of the functional organization of the same animal over time.The present study describes the fulfillment of both requirements in the rat primary somatosensory cortex. We focus on the representations of the mystacial vibrissae (whiskers), as this sensory system offers several unique structural and functional features. The pathways within the neuraxis that carry vibrissal information are well characterized both anatomically (1-4) and physiologically (1,3,(5)(6)(7). The whisker area of rat somatosensory cortex contains distinct cellular