This study examined whether glial cells in the trigeminal nucleus caudalis (Sp5C) were necessary for orofacial nociception and nociceptive processing induced by subcutaneously (s.c.) injection of 5% formalin into left mystacial vibrissae. The immunohistochemical, immunoelectron microscopical methods and behavior assessment were used in this study. Two hours after administration of carbenoxolone (CBX, a gap junction blocker) or fluorocistrate (FCA, a glail metabolic inhibitor) into the cerebellomedullary cistern, the nociceptive behavior and scratching-cumulative time reduced significantly (P<0.01). FCA attenuated obviously the expression of Fos/NeuN-immunoreactive (-IR) neurons (mean+/-S.E.M.=29+/-2.5) and Fos/glial fibrillary acidic protein (GFAP)-IR astrocytes (7.2+/-2.2) in Sp5C. CBX decreased the number of Fos/NeuN-IR neurons (25+/-1.7), but did not affect Fos/GFAP-IR astrocytes (16.2+/-5.4), compared with vehicle-preadministered rats (Fos/NeuN-IR neurons 135+/-4.2, and Fos/GFAP-IR astrocytes 25.8+/-4). Immunoelectron microscopy established that Cx32/Cx43 heterotypic gap junctions (HGJs) were present on junction areas between astrocytes and neurons within Sp5C. The number of HGJs increased significantly following formalin s.c. injection. It suggests that the Sp5C astrocytes may play an active regulating role in orofacial nociception via Cx32/Cx43 HGJs between astrocytes and neurons of Sp5C.