The inducible form of nitric oxide synthase (iNOS) generates nitric oxide of which the excessive production is associated with central nervous system (CNS) inflammatory diseases. The investigation of iNOS expression during experimental allergic encephalomyelitis (EAE) of the Lewis rat demonstrated iNOS immunoreactivity and mRNA both during inflammatory bursts (days 12 and 23 post-immunization) and during the remission phase (day 18). iNOS expression was region-specific and expanded with time along a caudo-rostral axis, thus, correlating with the development of inflammatory infiltrates. Whereas cells of the monocyte/macrophage lineage continuously contributed to iNOS expression, astrocytes only expressed iNOS immunoreactivity or mRNA during the relapse (day 23). In order to investigate possible regulatory effects of 1,25-dihydroxyvitamin D3 (1,25-D3) on iNOS expression, rats were treated with the hormone after the beginning of clinical signs (days 11, 13, 19, 21 and 23 post-immunization), and areas of the CNS were examined at day 23. 1,25-D3 exerted a drastic inhibitory effect on iNOS expression, both at the protein and the mRNA levels. However, this effect was region-specific, and was most pronounced in the cerebellum and brainstem, but non-existent in cerebral cortex. iNOS down-regulation occurred in macrophages, activated microglia and astrocytes. The inhibition of iNOS expression in some CNS structures could account for the improvement of clinical signs observed in EAE-rats treated with 1,25-D3. Since 1,25-D3 can be synthesized by activated macrophages or microglia, our results support the hypothesis that this hormone might be implicated in the control of the CNS-specific immune responses. 1,25-D3 or its analogues could, thus, be of therapeutic value in the management of iNOS-associated diseases of the CNS.