1,25-dihydroxyvitamin D3 (1,25-D3) is already known to prevent clinical signs of experimental allergic encephalomyelitis when animals are treated during the immunization phase. In the present work we have evaluated the ability of 1,25-D3 to inhibit chronic relapsing experimental allergic encephalomylitis (EAE) of the Lewis rat, when administered after the beginning of clinical signs. We observed a significant clinical improvement in 1,25-D3-treated rats. This effect was accompanied by a profound inhibition of CD4 antigen expression by central nervous system (CNS) infiltrating monocytes/macrophages and parenchymal microglia. In addition, immunohistochemical analysis performed at the time of the second attack evidenced a region-specific distribution of inflammatory cells. In the same way, some aspects of the effects exerted by 1,25-D3 appeared to vary depending on the region considered, namely spinal cord, brainstem, cerebellum, midbrain or anterior brain. Thus, in 1,25-D3-treated rats, we observed an almost complete inhibition of CD4 antigen expression in the granule cell layer and the adjacent white matter of the cerebellum as well as a marked decrease in the number of OX42-positive cells (macrophages and activated microglia) in anterior brain sections. We conclude that 1,25-D3 can exert immunomodulatory effects inside the CNS during an ongoing immune process and may thus represent a promising therapy for multiple sclerosis.