The functional organization of laryngeal motoneurons in the nucleus ambiguous (NA) was evaluated in adult male rats before and after recurrent laryngeal nerve section and reinnervation. Using retrograde double labeling techniques with fluorescent probes, we obtained the number and position of labeled neurons by using the Bioquant 3-D imaging system. Reinnervation was documented by electromyography. In nine control animals vector analysis revealed significant (p less than .05) separation of the posterior cricoarytenoid (PCA) muscle motoneurons and the thyroarytenoid and lateral cricoarytenoid (TA/LCA) muscle motoneurons. The PCA motoneurons were positioned ventromedially in the NA, and TA/LCA motoneurons were found dorsolaterally in the NA. Rostral-caudal separation was not significant. Electromyography revealed phasic electrical activity synchronous with respiration in the PCA, and activity synchronous with deglutition in the TA/LCA. In four animals surviving 15 weeks following recurrent laryngeal nerve section and primary neurorrhaphy, functional organization within the NA was lost and phasic motor unit activity synchronous with respiration was seen in the TA/LCA muscle as well as the PCA. Vector analysis revealed the reinnervating motoneurons for both the PCA and TA/LCA to be positioned dorsolaterally, similar to the control group TA/LCA motoneurons. These findings demonstrate a shift in the topographic organization of laryngeal motoneurons within the NA following reinnervation, with random organization occurring at the neurorrhaphy site.