The aim of this work was to study the ultrastructural distribution of substance P-like immunoreactivity in laminae I and II of rat spinal cord and trigeminal subnucleus caudalis in relation to synaptic glomeruli. A bispecific monoclonal antibody directed against substance P and horseradish peroxidase was used, combining sensitive immunocytochemistry with preservation of fine ultrastructural detail. Some of the quantitative observations were carried out with an automated image analysis system. The study revealed that in lamina I of the spinal cord, almost all immunoreactive profiles counted were nonglomerular, and a considerable number of them contacted medium-size or large dendrites or were in direct contact with other vesicle-containing profiles. In ventral lamina II, 9.4% of the labeled axonal varicosities were central boutons of type I glomeruli (CI). They could be identified by their scalloped contour, number and types of peripheral profiles, reduced density of mitochondria, and localization in the dorsal horn. However, these immunoreactive glomerular CI boutons (14.1% of the total number of CI) differed statistically from the prevailing population of nonimmunoreactive CI, by being surrounded by less peripheral neuronal profiles, which established fewer synapses. In addition, they contained more than three dense-core vesicles per central profile. In the trigeminal subnucleus caudalis laminae I and II, the substance P fibers and varicosities had a plexiform orientation at the light microscopic level, which contrasted with the mainly rostrocaudal orientation of the spinal cord's lamina II plexus. However, the main ultrastructural findings were similar. These results demonstrate that substance P-like immunoreactivity occurs in a large number of type I synaptic glomeruli with specific morphological features and reinforce the current concept that the substantia gelatinosa of the spinal cord and trigeminal subnucleus caudalis are homologous structures.