Interferon-β (IFN-β) exposure during tumour necrosis factor-α (TNF-α)-induced human monocyte-derived dendritic cell (DC) maturation augments the capacity of DC to promote the generation of T helper 1 (Th1) cells, while IFN-β exposure during naive Th cell stimulation inhibits Th1 cell generation (Nagai et al., J Immunol, 2003 171:5233–43). Investigating these contradictory outcomes of IFN-β exposure, we find that isolated DC matured with both TNF-α and IFN-β secrete more IL-12 p70 upon CD40L stimulation than DC matured with TNF-α alone. mAb blocking studies indicate that the basis for this enhanced IL-12 p70 production is augmentation of two successive CD40-dependent autocrine pathways in the DC: (1) a pathway in which low levels of IL-12 p70, IL-27, IL-18 and, possibly, IL-23 act to mediate autocrine induction of DC IFN-γ secretion; and (2) an IFN-γ-initiated autocrine pathway promoting optimal DC IL-12 p70 secretion. In contrast to the IL-12 p70 promoting effects of IFN-β during DC maturation, IFN-β pre-treatment before CD40L stimulation was found to inhibit IFN-γ-mediated enhancement of DC IL-12 p70 secretion. Thus, IFN-β exposure during TNF-α-mediated DC maturation may promote Th1 polarization by increasing DC IL-12 p70 secretion, through enhancement of autocrine-acting IFN-γ production by the DC. Moreover, IFN-β exposure during naive Th cell stimulation may inhibit Th1 cell generation by blocking the IFN-γ-induced signals required for optimal CD40L-induced DC IL-12 p70 secretion. IFN-β pre-treatment was also observed to inhibit CD40L-induced DC IL-23 secretion. Our findings may account for some of the beneficial effects of IFN-β therapy in patients with relapsing remitting multiple sclerosis.