1. The ability of rats to localize sounds in space was determined before and after cutting of the commissure of Probst. The commissure of Probst was transected at its midline decussation with a microknife inserted into the brain according to stereotaxic coordinates. Six animals were tested after extensive lesions that destroyed all of the commissure of Probst fibers. An additional animal was tested after a smaller lesion that destroyed most of the commissure of Probst but left some fibers intact. Three control animals were tested before and after surgical intervention that did not involve the commissure of Probst. 2. The animals were tested in a semicircular apparatus with loudspeakers located on the right or left of midline. They were trained to make a response toward the left or right in the direction of the active loudspeaker. Correct reponses were rewarded by delivery of a small quantity of water from spouts located at +30 and -30 degrees azimuth. Tests of sound localization were conducted with a single broadband noise burst, 45 ms in duration, presented at the beginning of each trial. The position of the active loudspeakers was varied from trial to trial and performance at different speaker angles was calculated to determine psychometric curves. Minimum audible angles were estimated by interpolation from a performance level of 75% correct. 3. After postoperative testing was completed, the effectiveness of the lesions was confirmed by cell counts to determine the extent of retrograde degeneration in the dorsal nucleus of the lateral lemniscus (DNLL). These data showed that most of the contralaterally projecting neurons in DNLL underwent retrograde degeneration and the number of neurons was reduced by 60-65%. Ninety to 95% of the contralaterally projecting neurons in the DNLL disappeared within 7 wk after transection of the commissure of Probst. 4. The condition of damaged commissural fibers was further confirmed by tract tracing methods. A unilateral Fluoro-Gold injection was made into the inferior colliculus and the auditory brain stem was examined for retrograde label. No labeled neurons were seen in the contralateral DNLL in cases with complete transection of the commissure of Probst. In addition, the distribution of Fluoro-Gold labeling in other brain stem auditory structures was similar to that seen in normal animals. This result confirmed that the transection of the commissure of Probst was successful and that projections to other auditory structures remained intact. 5. Transection of the commissure of Probst produced marked deficits in midline sound localization. Although sound localization was still possible, there was a degradation in the ability of rats to localize sounds in the horizontal plane. The mean minimum audible angle was elevated 22.1 degrees after destruction of the commissure of Probst compared with a shift of only 2.0 degrees in control animals.