Our previous studies have indicated a critical role of protein kinase C (PKC) in intracellular mechanisms of tolerance to morphine analgesia. In the present experiments, we examined (1) the cellular distribution of a PKC isoform (PKC gamma) in the spinal cord dorsal horn of rats associated with morphine tolerance by utilizing an immunocytochemical method and (2) the effects of the N-methyl-D-aspartate receptor antagonist MK-801 on tolerance-associated PKC gamma changes. In association with the development of tolerance to morphine analgesia induced by once daily intrathecal administration of 10 micrograms morphine for eight days, PKC gamma immunoreactivity was clearly increased in the spinal cord dorsal horn of these same rats. Within the spinal cord dorsal horn of morphine tolerant rats, there were significantly more PKC gamma immunostained neurons in laminae I-II than in laminae III-IV and V-VI. Such PKC gamma immunostaining was observed primarily in neuronal somata indicating a postsynaptic site of PKC gamma increases. Moreover, both the development of morphine tolerance and the increase in PKC gamma immunoreactivity were prevented by co-administration of morphine with 10 nmol MK-801 between Day 2 and Day 7 of the eight day treatment schedule. In contrast, PKC gamma immunoreactivity was not increased in rats receiving a single i.t. administration of 10 micrograms morphine on Day 8, nor did repeated treatment with 10 nmol MK-801 alone change baseline levels of PKC gamma immunoreactivity. These results provide further evidence for the involvement of PKC in NMDA receptor-mediated mechanisms of morphine tolerance.