A previous study has shown that mesencephalic periodontal afferent terminals receive contacts more frequently from axonal endings containing pleomorphic, synaptic vesicles (P-endings) in the supratrigeminal nucleus (Vsup) than in the trigeminal motor nucleus, suggesting that interneurons in Vsup play an important role in modulating the jaw-closing reflex. The present study was attempted to identify neurotransmitters in P-endings associated with mesencephalic periodontal afferents in cat Vsup through the use of intracellular staining of horseradish peroxidase combined with the postembedding immunogold methods. A morphometric analysis was carried out to compare the ultrastructural features of these two types of terminals. Serial sections of 31 labeled boutons and of their associated 38 P-endings were examined. They were processed for postembedding immunogold labeling with antibodies to the neurotransmitter gamma-aminobutyric acid (GABA). The 38 P-endings presynaptic to periodontal afferents showed GABA-like immunoreactivity, but the afferent terminals were free from the labeling. The morphometric analysis indicated that bouton volume, apposed surface area, total active zone size, and mitochondrial volume were smaller in GABA-immunoreactive P-endings than in periodontal afferents, but the pooled data of the two types of terminals showed that each synaptic parameter was highly correlated in a positive, linear manner with bouton volume. These observations provide evidence that P-endings presynaptic to mesencephalic periodontal afferents contain the neurotransmitter GABA and that their axoaxonic synapses are organized in accordance with the ultrastructural "size principle" proposed by Pierce and Mendell (Pierce and Mendell [1993] J. Neurosci. 13:4748-4763) on Ia-motoneuron synapses.