The relationship between retrogradely labelled or intracellularly filled phrenic motoneurons and varicosities containing thyrotropin-releasing hormone immunoreactivity was investigated in rats by light and electron microscopy. Phrenic motoneurons were identified via retrograde tracing from the diaphragm with cholera toxin B subunit, which was followed by immunocytochemistry to visualise retrogradely labelled motoneurons and thyrotropin-releasing hormone-immunoreactive nerve fibres in their vicinity. At the light microscopic level, varicose thyrotropin-releasing hormone-immunoreactive nerve fibres were distributed sparsely in the phrenic motor nucleus, with some axons surrounding retrogradely labelled motoneurons. In separate intracellular experiments, four phrenic motoneurons identified by antidromic activation from the C5 phrenic nerve root were subsequently filled with Neurobiotin, and nerve fibres that contained thyrotropin-releasing hormone immunoreactivity were identified by immunocytochemistry. The numbers and locations of thyrotropin-releasing hormone-immunoreactive varicosities that were closely appeared to the intracellularly labelled motoneurons were mapped using a camera lucida technique. Close appositions by thyrotropin-releasing hormone-immunoreactive varicosities were seen on somata as well as on proximal and distal dendrites. The closely apposed varicosities were usually present in tight clusters, which were formed by single varicose axons. However, the distribution was nonuniform, in that some dendrites did not receive any close appositions. Ultrastructural analysis of random ultrathin sections through retrogradely labelled neurons showed that varicosities with thyrotropin-releasing hormone immunoreactivity made 1.8% of all synapses and direct contacts on somata and 2.3% of synapses and contacts with dendrites of the retrogradely labelled phrenic motoneurons. The results of these experiments suggest that thyrotropin-releasing hormone-immunoreactive varicosities provide similar numbers of inputs to both the somata and dendrites of phrenic motoneurons. These thyrotropin-releasing hormone-containing inputs seen via light and electron microscopy could modulate the excitability of phrenic motoneurons.