Objective: We investigated dopaminergic inhibition of CD4+CD25high regulatory T lymphocytes (Treg) in relapsing-remitting multiple sclerosis (MS) patients treated with interferon (IFN)-β. Methods: MS patients were prospectively studied at baseline and during 1 year of IFN-β, and compared with healthy controls (HCs). Treg were separated by immunomagnetic sorting and the effect of dopamine (DA) on Treg was assessed in coculture experiments with homologous effector T lymphocytes (Teff). Tyrosine hydroxylase (TH), dopaminergic receptors (DR) D3 and D5, and forkhead box protein P3 (FoxP3) mRNA were assessed by real-time PCR. Circulating CD4+ T cell subsets were assessed by flow cytometry. Results: In coculture experiments, Treg inhibition of Teff proliferation was reduced by DA in HCs and completely abolished in MS patients at baseline. However, in patients after 12 months of IFN-β, Teff proliferation was impaired and DA had no more effect on Treg. In comparison to cells from HCs, Treg from MS patients at baseline had increased mRNA for DR D5 and TH (but not for DR D3). During treatment with IFN-β, both DR D5 and TH mRNA decreased down to values lower than those of cells from HCs. In comparison to HCs, MS patients had a higher frequency of circulating Treg, both at baseline and after IFN-β, while FoxP3 mRNA levels in Treg were similar in patients and HCs and did not show major changes during IFN-β. Conclusions: Dopaminergic inhibition of Treg in MS patients is suppressed during IFN-β treatment. Treg play a key role in the suppression of autoimmunity, thus the effect may have a therapeutic repercussion.