The congenital absence of the corpus callosum, a brain anomaly frequently noted in humans, has been recently found to occur in some mice of the ddN strain in our laboratory. In the brains of these mice, the Probst's longitudinal bundle is always present on both cerebral hemispheres and gives rise to some aberrant fibers toward the midline. In this research, the neuroanatomical features of these fibers were studied by iontophoretical injections of horseradish peroxidase (HRP) into the neocortex of acallosal mouse brains. The results revealed that the fibers which leave the Probst's longitudinal bundle are, at least, of 3 kinds: namely, the fibers that run out from the anterior portion of the bundle and take a U-turn ipsilaterally without crossing the midline through the septal tissue to go back again into the longitudinal bundle at the level where they have left it; the commissural fibers that leave the bundle from its middle portion and cross through a tiny bridge of tissue associated with the ventral hippocampal commissure to the opposite hemisphere; and the fibers that arise from the posterior portion of the bundle and accumulate as an anomalous fascicle below the cingulum. The observation that no labeled fibers were seen within the anterior commissure in the present HRP materials suggests that the axons from neocortex which are prevented from crossing the midline in mice with congenital absence of the corpus callosum cannot find an alternative pathway via the anterior commissure.