Our previous observations have shown that neuroactive steroid levels in the brain are affected by acute experimental autoimmune encephalomyelitis (EAE) with sex and regional specificity (Giatti et al. 2010). To better understand the effect of EAE on neuroactive steroids, we have here assessed the levels of pregnenolone, progesterone and its derivatives (i.e. dihydroprogesterone, tetrahydroprogesterone and isopregnanolone), testosterone and its derivatives (dihydrotestosterone and 5alpha-androstane-3alpha, 17beta-diol) in different CNS regions of male and female rats affected by chronic EAE. Data obtained by liquid chromatography tandem mass spectrometry revealed that chronic EAE results in sex and regional specific alterations in the levels of neuroactive steroids in the brain, which are in many cases different to those produced by acute EAE. The specific changes in neuroactive steroid levels after chronic EAE may be of relevance to design new possible therapeutic strategies for the disease.