Predominance of multiple sclerosis (MS) in women, reductions of disease flares during pregnancy, and their increase in the postpartum period have suggested a hormonal influence on MS activity. The hormone prolactin (PRL) has long been debated as a potential immune-stimulating factor in several autoimmune disorders, including MS and its animal model experimental autoimmune encephalomyelitis (EAE). However, to date, no data clearly ascribe a pathogenic role to PRL in these diseases. Using PRL receptor-deficient (Prlr(-/-)) and PRL-deficient (Prl(-/-)) mice, we show that PRL plays a redundant role in the development of chronic EAE. In Prlr(-/-) and Prl(-/-) mice, EAE developed with a delayed onset compared with littermate control mice, but with full clinical severity. In line with the clinical outcome, T cell proliferation and production of IFN-γ, IL-17A, and IL-6 induced by myelin Ag were delayed in Prlr(-/-) and Prl(-/-) mice. Ag-specific IgG Ab responses were not affected by PRLR or PRL deficiency. We also show that mouse lymph node cells and purified CD4(+) T cells express transcript for Prlr, but not for Prl. These results reveal that PRL does not play a central role in the development of chronic EAE and optimal Th1 and Th17 responses against myelin. Moreover, they also rule out a possible contribution of PRL secreted by immune cells to the modulation of autoreactive T cell response in this model.