Acute and chronic peripheral inflammation is known to induce the expression of cyclo-oxygenase (COX)-2 in spinal cord neurons and increase the synthesis and release of prostaglandins (PG). Although these PG are presumed to cause inflammatory pain or hyperalgesia, the relationship between PG-producing cells in the dorsal horn and substance P (SP)-containing, pain-transmittimg nerve fibers remains unknown. In the present study we investigated immunohistochemically changes in the number of COX-2-containing neurons using the avidin-biotinylated peroxidase complex method in dorsal horn superficial laminae in chicken lumbosacral enlargement (L4, L5) under inflammatory conditions induced by unilateral intraplantar injection of complete Freund's adjuvant. After 12-24 h, a significant increase in the number of small COX-2-containing neurons was observed in lamina II on the injected side compared with the contralateral side. Furthermore, using fluorescent double-labeling for COX-2 and SP, an increase in the number of small COX-2-containing neurons in contact with SP-containing elements was observed ipsilaterally (1.4-1.6-fold compared with the contralateral side) in lamina II. Fluorescence triple-labeling of COX-2, SP and calcitonin gene-related peptide (CGRP) confirmed that the majority of these SP-containing elements coexisted with CGRP, indicating that these elements originated from primary afferent neurons. Using electron microscopy, two types of SP-containing axon terminals were found to form synapses with COX-2-containing neurons in lamina II. These results indicate that the number of COX-2-containing neurons increases concomitantly with an increase in the number of contacts of these neurons with SP-containing primary afferent fibers and suggest that this phenomenon is associated with PG production and the persistence of inflammatory pain.