The functional specificity of callosal connections was investigated in visual areas 17 and 18 of adult cats, by combining in vivo optical imaging of intrinsic signals with labeling of callosal axons. Local injections of neuronal tracers were performed in one hemisphere and eight single callosal axons were reconstructed in the opposite hemisphere. The distributions of injection sites and callosal axon terminals were analyzed with respect to functional maps in both hemispheres. Typically, each callosal axon displayed 2 or 3 clusters of synaptic boutons in layer II/III and the upper part of layer IV. These clusters were preferentially distributed in regions representing the same orientation and the same visuotopic location as that at the corresponding injection sites in the opposite hemisphere. The spatial distribution of these clusters was elongated and its main axis correlated well with the preferred orientation at the injection site. These results demonstrate a specific organization of interhemispheric axons that link cortical regions representing the same orientation and the same location of visual stimuli. Visual callosal connections are thus likely involved in the processing of coherent information in terms of shape and position along the midline of the visual field, which may facilitate the fusion of both hemifields into the percept of a single visual scene.