The posterior cricoarytenoid (PCA) muscle is the sole abductor of the larynx and is controlled by motoneurons located in the nucleus ambiguus. These motoneurons receive inputs from a variety of interneurons, including those that impart respiratory modulation, and are responsible for the phasic inspiratory activity of the PCA muscle. Identification of PCA motoneurons is therefore an essential initial step in understanding the mechanisms responsible for coordinated vocal cord abduction. We identified PCA motoneurons in the rat model by retrograde labeling, and following antidromic activation. A total of 194 neurons were identified by retrograde labeling with cholera toxin B subunit (CTB). Labeling was exclusively ipsilateral where the contralateral vagus and superior laryngeal nerves had been divided. The neurons were multipolar, with dimensions of 33.2 +/- 6.4 microm (mean +/- standard deviation) in length and 22.4 +/- 3.4 microm in width. The neurons were located within a range of 0.6 to 2.4 mm caudal to the caudal pole of the facial nerve, 1.2 to 1.7 mm lateral to the midline, and 1.5 to 2.3 mm deep to the dorsal surface of the medulla. The PCA motoneurons were antidromically activated by focal stimulation of the PCA muscle. The extracellular field was recorded in 5 rats, and the PCA motoneurons were found within a range of 0.8 to 1.7 mm caudal to the caudal pole of the facial nerve, 1.5 to 2.0 mm lateral to the midline, and 1.9 to 2.4 mm deep to the dorsal surface of the medulla. The mean conduction velocity ranged from 37.0 +/- 5.8 to 68.6 +/- 5.0 m/s. An extracellular antidromic field potential, which corresponds to the distribution of the PCA motoneuron pool demonstrated by retrograde labeling with CTB, can be reliably obtained in a rat model following focal PCA muscle stimulation.