1. The rat whisker-to-barrel system was used to investigate the variability and interhemispheric asymmetry in the functional organization of primary somatosensory cortex as assessed with intrinsic signal optical imaging. The areal extent of whisker D1 functional representation was determined for both the left and right barrel cortex of each of 10 adult male rats. The average size of whisker D1 functional representation and the amount of variability away from this average across animals were determined. In addition, interhemispheric asymmetry was addressed at both the population level and the individual level. The degree of side preference for thigmotactic scanning (typical whisker-related rodent behavior) was determined for each rat in an attempt to find a behavioral correlate for the degree of interhemispheric asymmetry in the size of whisker D1 functional representation. 2. The average areal extent of whisker D1 functional representation (defined as area at half-height) was large (1.95 +/- 0.14 mm2, mean +/- SE, N = 10 rats), suggesting that stimulation of a single whisker evokes activity over a large cortical area that includes other whisker representations. 3. The average size of whisker D1 functional representation was not significantly different between the left (1.86 +/- 0.21 mm2) and right (2.04 +/- 0.15 mm2) hemispheric side, suggesting that interhemispheric functional asymmetry of barrel cortex is not systematic toward a specific hemispheric side at the population level. 4. The degree of variability in the size of whisker D1 functional representation from the left hemisphere ranged between 54.6% smaller than to 50.6% larger than the left average areal extent. A large degree of variability was also observed for the right D1 representation, 37.6% smaller than to 34.9% larger than the right average areal extent. Thus it appears that a large variability in the size of unmanipulated single-whisker functional representations exists across animals from the same species and is not exclusive to a particular hemispheric side. 5. In 5 of 10 rats, the size of whisker D1 functional representation between the two hemispheres differed by > or = 25% within an individual animal. Of these five rats, four had a larger representation in their right hemisphere. The degree and direction of behavioral asymmetry was not linearly correlated with the interhemispheric asymmetry in the size of D1 functional representation (r = 0.494). 6. The large size of a single-whisker functional representation as defined with intrinsic signal optical imaging is discussed with respect to previous anatomic and 2-deoxyglucose autoradiography studies, whereas the large variability in this size across animals is discussed with respect to the individuality of each animal. In addition, the results of the present study have implications for projects that plan to investigate relative changes in the size of single-whisker functional representations.