Unilateral hindpaw inflammation produces several neurochemical changes in the ipsilateral spinal dorsal horn that have been interpreted as contributing to the associated hyperalgesia. NADPH-diaphorase histochemical staining was used to examine the response of a population of neurons 1, 2, 6 or 24 h following injection of 6 mg carrageenan into the left hindpaw of the rat. The resulting unilateral hindpaw inflammation produced a bilateral, time-dependent, reversible increase in the number of NADPH-diaphorase stained neurons in the lumbar spinal cord that peaked at 6 h. In laminae I-III, there was a significant increase in the number of NADPH-diaphorase stained neurons both ipsilateral (25.9 +/- 2.3) and contralateral (26.3 +/- 1.3) to the inflamed hindpaw relative to uninflamed, control animals (18.6 +/- 1.7, 17.4 +/- 1.7, respectively). A smaller but significant increase was observed in laminae IV-VII and X. Under dark field illumination, an increase in the number of densely stained neurons in laminae I-III was also observed to peak at 6 h. A greater percentage of the neurons observed under bright field illumination were visible under dark field illumination at 6 h (47%) compared to control (18%), suggesting an increase in the enzymatic activity of neurons in laminae I-III in addition to the increase in the number of neurons with threshold levels of NADPH-diaphorase activity. There was no consistent increase in this ratio over time in laminae IV-VII or X. Six hours following carrageenan, there was a bilateral 50% increase in the density of NADPH-diaphorase staining in the neuropil in the medial laminae I-III. Both spinal neurons and primary afferent axons contributed to this bilateral increase in staining as the number of NADPH-diaphorase stained dorsal root ganglion cell bodies increased 47% over control. In addition to the increase in staining in the lumbar spinal cord, at 6 h post carrageenan, there was a bilateral 23% increase over control in the number of stained neurons in the cervical dorsal spinal cord. For comparative purposes, the distribution of Fos-immunoreactive nuclei was compared to NADPH-diaphorase 6 h post carrageenan. The distribution of Fos-immunoreactive nuclei differed from the NADPH-diaphorase stained neurons, suggesting that two separate populations of neurons were stained. Unilateral hindpaw inflammation did not result in an increase in NADPH-diaphorase activity in the periaqueductal gray or the ventroposteriolateral thalamic nucleus. The relationship of NADPH-diaphorase to nitric oxide is discussed and it is concluded that NADPH-diaphorase,(ABSTRACT TRUNCATED AT 400 WORDS)