Expression of nitric oxide synthase (NOS) was investigated in neurons of lumbar spinal cord of adult rats following subcutaneous injection of formalin (FOR) in one hindpaw. NOS was visualized immunocytochemically using a specific antibody and by the NADPH-diaphorase reaction (NDP). In the untreated rat, NOS immunoreactivity (IR) and NDP were present in neurons of the superficial dorsal horn (sDH) predominantly in layers II-III, and in the deep dorsal horn (dDH) predominantly in layer X. Twenty-four hours following FOR, the numbers of neurons labelled for NOS and NDP and the density of NDP containing nerve fiber varicosities significantly increased in sDH of the ipsilateral L3-L4 segments. NOS-IR and NDP gave a rather congruent distribution of labelled neurons in the dorsal horn. In contrast, distinct NOS-IR but not NDP was visible in large diameter motoneurons and in the lateral spinal nucleus. Double labelling demonstrated that in sDH most of the NDP-reactive neurons show a close spatial relationship to fibers and varicosities immunoreactive for substance P and CGRP. These neuropeptides are considered mediators of synaptic input from nociceptive primary afferents. Colocalization of NDP with c-Jun, JunB, JunD, c-Fos, FosB and Krox-24 transcription factors was investigated in neurons of lumbar spinal cord. c-Jun, JunB, c-Fos and Krox-24 reached their maximal levels of expression 2 h after FOR and returned to basal levels after 10 h. FosB and JunD reached their maximal expression after 5 h, persisted up to 10 h and were still visible in 60%-70% of the maximal number of labelled nuclei after 24 h. This persistent expression of transcription factors might contribute to the up-regulation of NOS expression between 10 h and 24 h. In a low number of NDP neurons, suprabasal immunoreactivity of JunB, c-Fos and Krox-24 proteins was visible up to 10 h, and of JunD and FosB up to 24 h in sDH neurons; c-Jun was not expressed in NDP labelled neurons of sDH, but, similar as JunD, showed basal colocalization in preganglionic sympathetic and parasympathetic neurons. In dDH, colocalization of Jun, Fos and Krox-24 proteins in few neurons was only observed following a second FOR stimulus given 24 h after the first one. Double-staining also demonstrated that many Jun, Fos and Krox labelled neurons are in close proximity to NDP labelled nerve fibers suggesting a functional relationship between expression of immediate-early gene encoded transcription factors and presence of nitric oxide in the rat spinal cord.