Although treatment of multiple sclerosis (MS) with the type I interferon (IFN) IFN-β lowers disease activity, the role of endogenous type I IFN in MS remains controversial. We studied CD4+ T cells and CD4+ T cell subsets, monocytes and dendritic cells by flow cytometry and analysed the relationship with endogenous type I IFN-like activity, the effect of IFN-β therapy, and clinical and magnetic resonance imaging (MRI) disease activity in MS patients. Endogenous type I IFN activity was associated with decreased expression of the integrin subunit CD49d (VLA-4) on CD4+CD26(high) T cells (Th1 helper cells), and this effect was associated with less MRI disease activity. IFN-β therapy reduced CD49d expression on CD4+CD26(high) T cells, and the percentage of CD4+CD26(high) T cells that were CD49d(high) correlated with clinical and MRI disease activity in patients treated with IFN-β. Treatment with IFN-β also increased the percentage of CD4+ T cells expressing CD71 and HLA-DR (activated T cells), and this was associated with an increased risk of clinical disease activity. In contrast, induction of CD71 and HLA-DR was not observed in untreated MS patients with evidence of endogenous type IFN I activity. In conclusion, the effects of IFN-β treatment and endogenous type I IFN activity on VLA-4 expression are similar and associated with control of disease activity. However, immune-activating effects of treatment with IFN-β may counteract the beneficial effects of treatment and cause an insufficient response to therapy.