An experimental arthritis induced by injection of kaolin and carrageenan into the knee joint resulted in a temporal relationship between glutamate dorsal horn content and paw withdrawal latency (PWL) which was positively correlated. Limping, guarding, increased response to heat stimuli (hyperalgesia) and altered staining patterns for glutamate (GLU), substance P (SP), and calcitonin gene-related peptide (CGRP) were monitored in the awake behaving arthritic rat over a 1 week time course. A decrease in PWL occurred on the side ipsilateral to the inflamed knee as early as 4 h after the induction of arthritis indicating the animals are hyperalgesic. The PWL remained decreased through the first 24 h. Computer-assisted quantification of the density of immunohistochemical staining indicated the content of GLU, SP and CGRP was altered differentially throughout the time course of the arthritis. The changes observed for all three substances occurred across the entire superficial dorsal horn. There was an initial depletion of SP followed by an increase in both SP and CGRP content which was maintained through 1 week. The GLU content was increased during the hyperalgesic period. The GLU changes followed the same time course and were positively correlated with the changes in PWL. In a small group of animals injected with kaolin and carrageenan, hyperalgesia did not develop. In this group of animals, no change in dorsal horn GLU or SP content occurred. Rather, there was an increase in CGRP content in the middle portion of the superficial dorsal horn which is the termination site of knee joint afferents. These data indicate that the development of heat hyperalgesia is dependent on GLU and possibly SP. Since inflammation of the knee joint does not involve the foot pad, the heat hyperalgesia observed during the first 24 h following induction of arthritis represents a central neuronal sensitization.