Treatment with interferon (IFN)-β reduces clinical disease activity in multiple sclerosis (MS). Using flow cytometry, an enzyme-linked immunosorbent assay and a real-time polymerase chain reaction, we studied in vivo IFN-β-induced effects on CD4+ T-lymphocyte chemokine receptor expression as these influence central nervous system (CNS) transmigration and inflammation. At ‘steady state’ (≥1 day after the most recent IFN-β injection), IFN-β treatment increased CD4+ T-cell surface expression of CC chemokine receptor (CCR)4, CCR5 and CCR7 after 3 months of treatment, whereas that of CXC chemokine receptor (CXCR)3 was unaltered. Conversely, at 9–12 h after the most recent IFN-β injection, CCR4, CCR5 and CCR7 expressions were unaltered, while CXCR3 expression was reduced. CD4+ T-cell surface expression of CCR4 was significantly lower in untreated MS patients compared with healthy volunteers. Of the plasma chemokines, only CXCL10 was increased by IFN-β treatment; CCL3, CCL4, CCL5 and CXCL9 were unaltered. CCR5 mRNA expression in blood mononuclear cells correlated with the expression of T-helper type 1 (Th1)-associated genes whereas CCR4 and CCR7 mRNA expression correlated with Th2 and immunoregulatory genes. In conclusion, IFN-β treatment caused ‘steady-state’ increases of several chemokine receptors relevant for CD4+ T-lymphocyte trafficking and function, possibly facilitating lymphocyte migration into the CNS. An important therapeutic effect of IFN-β treatment may be the normalization of a decreased Th2-related CD4+ T-cell CCR4 expression in MS patients. Surface chemokine receptor expression and CXCL10 varied according to the timing of blood sampling in relation to the most recent IFN-β injection. Thus, it is imperative to distinguish acute effects of IFN-β from steady-state effects.