In the present study, we assessed the extent of the thyrotropin-releasing hormone (TRH) input to motoneurons in the ambigual, facial, and hypoglossal nuclei of the rat using a combination of intracellular recording, dye filling, and immunohistochemistry. Twelve motoneurons in the rostral nucleus ambiguus were labelled by intracellular injection in vivo of Neurobiotin (Vector). Seven out of 12 ambigual motoneurons displayed rhythmic fluctuations of their membrane potential in phase with phrenic nerve discharge, whereas the other five had no modulations of any kind. Seven facial motoneurons and seven hypoglossal motoneurons were also filled with Neurobiotin. All three motor nuclei contained TRH-immunoreactive varicosities, with the largest numbers found in the nucleus ambiguus. Close appositions were seen between TRH-immunoreactive boutons and every labelled motoneuron. Respiratory-related motoneurons in the nucleus ambiguus received the largest number of TRH appositions with 74 +/- 38 appositions/neuron (mean +/- S.D.; n = 7). In contrast, nonrespiratory ambigual motoneurons received significantly fewer TRH appositions (11 +/- 5; n = 5; P < 0.05; Mann-Whitney U test). Facial motoneurons received about the same number of TRH appositions as nonrespiratory ambigual motoneurons, with 13 +/- 4 (n = 7). Hypoglossal motoneurons received the fewest appositions from TRH-containing boutons, with 8 +/- 2 (n = 7). There were no differences in the TRH inputs to respiratory and nonrespiratory motoneurons in the facial and hypoglossal nuclei. These results demonstrate that, among motoneurons in the medulla, respiratory motoneurons in the rostral nucleus ambiguus are preferentially innervated by the TRH-immunoreactive boutons.