The activity and expression of nitric oxide synthase (NOS) isoforms and protein nitrotyrosine (NT) residues were investigated in whole encephalic mass (WEM) homogenates during the development of experimental allergic encephalomyelitis (EAE) in Lewis rats. EAE stages (0-III) were daily defined by clinical evaluation, and in the end of each stage, WEMs were removed for analysis of NOS activity, protein NT residues and mRNA for the different NOS isoforms. In the presence of NADPH, WEMs from EAE-III rats showed lower Ca2+-dependent NOS activity than those from control group. These differences disappeared in the presence of exogenous calmodulin, flavin adenine dinucleotide (FAD), tetrahydrobiopterin (BH4) and NADPH. Of all the cofactors, just the omission of FAD caused comparable decrease of Ca2+-dependent NOS activity from both groups. Ca2+-independent NOS activity from EAE-III animals was insensitive to the omission of any of the cofactors, while in control animals this activity was significantly inhibited by the omission of either FAD or BH4. Increased levels of both iNOS mRNA and protein NT expression were observed in animals with EAE, which also showed lower levels of a thermolabile NOS inhibitor in WEM homogenates and sera than controls. In conclusion, during late EAE stages, constitutive Ca2+-dependent NOS activity decreases concomitantly with iNOS upregulation, which could be responsible for the high protein NT levels. The differential dependence of iNOS activity on cofactors and the absence of an endogenous thermolabile NOS inhibitor in animals with EAE could reflect additional control mechanisms of NOS activity in this model of multiple sclerosis.