Interferon (IFN)-β reduces the biological activity of multiple sclerosis (MS), a presumably T cell-mediated autoimmune disease of central nervous system (CNS) myelin. Co-stimulatory molecules are necessary for full T cell activation and differential expression of co-stimulatory molecules on antigen-presenting cells is thought to influence the type of effector T cell response (Th1/Th2). In this study we investigated the effects of IFN-β on the expression of co-stimulatory molecules on lymphocytes and monocytes as a potential mechanism of action of IFN-β in MS. Peripheral blood mononuclear cells (PBMCs) were stimulated with IFN-β in vitro and expression of CD80, CD86, CD40 and HLA was examined by flow cytometry and reverse-transcription polymerase chain reaction. Whereas IFN-β had no effect on the expression of these molecules on T and B lymphocytes there was a significant increase on monocytes. Correspondingly, the expression of mRNA increased after 6–18 h. This in vitro response was also observed in untreated MS patients and patients receiving treatment with IFN-β. The increase of co-stimulatory molecules on monocytes was not mediated by interleukin (IL)-10. When IFN-β-stimulated monocytes were used to stimulate autologous T cells an increased secretion of IL-13 was observed. In biopsies taken from IFN-β-induced skin reactions after subcutaneous injection increased expression of CD80 mRNA was detected, indicating that IFN-β also up-regulates this co-stimulatory molecule in vivo. These data provide the background for further studies of IFN-β-induced changes of co-stimulatory molecules in MS patients.