The aim of the present study was to examine the effect of intrathecal (i.t.) injection of pertussis toxin (PTX) on the nociceptive threshold and protein kinase C (PKC) expression in the rat spinal cord. The role of N-methyl-D-aspartic acid (NMDA) receptors in these changes was also examined. Male Wistar rats were implanted with two i.t. catheters, one of which was connected to a mini-osmotic pump and used to infuse saline or D-2-amino-5-phosphonopentanoic acid (D-AP5) (2 microg/h) starting on day 3 after i.t. catheter insertion. Two days later, a single injection of saline or PTX (2 microg) was given via the other catheter, followed by a flush with 10 microl of saline. On day 4 after PTX or saline injection, the thermal paw withdrawal latency was measured, then the rats were sacrificed by decapitation, and the dorsal part of the lumbosacral spinal segments was removed for PKC Western blotting assays. In PTX-treated rats, thermal hyperalgesia was observed, and the PKCgamma content of both the synaptosomal membrane and cytosolic fractions was significantly increased. The levels of alpha-, betaI-, or betaII-PKC isozymes in these fractions were unaffected by PTX treatment. Infusion of the NMDA antagonist, D-AP5, prevented both the thermal hyperalgesia and the increase in PKCgamma isoform expression in PTX-treated rats, and had no effect on these values in nai;ve rats. Intrathecal injection of the PKC inhibitor, chelerythrine (10 microg), significantly inhibited the thermal hyperalgesia observed in PTX-treated rats. These results show that i.t. injection of PTX induced thermal hyperalgesia accompanied by a selective increase in PKCgamma expression in both the synaptosomal membrane and cytosolic fractions of the dorsal horn of the rat lumbar spinal cord, and both effects were inhibited by the NMDA receptor antagonist, D-AP5.