Neonatal rats produce ultrasonic vocalizations in response to cold stress. The rate and intensity of these vocalizations decrease dramatically as the pup reaches maturity. The laryngeal nerves controlling the production of ultrasounds and their nuclei of origin were investigated in 10-day old rat pups. Unilateral and bilateral transections of the inferior laryngeal nerve reduced ultrasounds to undetectable levels. Transecting the superior laryngeal nerve either unilaterally or bilaterally reduced the sound pressure level, reduced the rate, and increased the fundamental frequency of the ultrasounds. Retrograde transport of horseradish peroxidase revealed that the efferent axons in the inferior laryngeal nerve arise from the cells of the dorsal formation of the nucleus ambiguus and that efferents in the superior laryngeal nerve originate from the ventral formation of the nucleus ambiguus. Therefore, different aspects of the production of pup ultrasounds appear to be controlled by distinct neuronal subpopulations of the nucleus ambiguus.