Three-dimensional (3-D) reconstruction of autoradiograms can provide new insights into the functional relationship of neural regions. To reach full potential, however, 3-D reconstruction must be both accurate and efficient. In this paper, we present a novel image matching algorithm that simultaneously aligns a set of serial sections and uses the method to reconstruct whisker barrels from the rat cerebral cortex. We initially compared several alignment techniques and found that our Multi-Set Registration (MSR) algorithm produced superior accuracy. This algorithm is based on a least-squares minimization technique and is able to simultaneously register a set of serial sections with subpixel precision (30-micron accuracy). We applied our new technique to the 3-D reconstruction of a series of autoradiograms. Our objective was to visualize and measure the 3-D metabolic (functional) shape of normal (control) and developmentally altered (plastic) C3 vibrissa columns in the first somatosensory area of the rat cerebral cortex. The plastic C3 metabolic column showed a nearly 450% increase in volume when compared to the control column. In addition, the lesion-altered C3 column-in contrast to the normal C3 column-displayed no central zone of high activity, and patches of higher metabolic activity were scattered throughout the columnar profile. This metabolic activity was not confined to the cylindrical column, but extended tangentially as radiating fingerlike projections toward neighboring barrels.