Division of Immunology and Allergy, Clinical Immunology Unit, Faculty of Medicine, University Hospital, Geneva, Switzerland. IFN-beta induces the production of secreted IL-1R antagonist (sIL-1Ra) without triggering synthesis of the agonist IL-1beta in human monocytes. This might account for its anti-inflammatory properties. Canonically, IFN-beta signals through activation of JAK/STAT pathway, although PI3K and MAPK have also been involved. In this study, the role of PI3K, MEK1, and STAT1 in IFN-beta-induced sIL-1Ra production is investigated in freshly isolated human blood monocytes. PI3K, but not MEK1 activation is essential for sIL-1Ra production in monocytes treated with IFN-beta, as demonstrated by using the respective inhibitors of PI3K and MEK1, Ly294002 and PD98059. The use of cycloheximide and actinomycin D shows that sIL-1Ra was an immediate early gene induced by IFN-beta and that PI3K was controlling sIL-1Ra gene transcription. Although both inhibitors of PI3K and MEK1 diminished the Ser(727) phosphorylation of STAT1 induced by IFN-beta, only Ly294002 inhibited sIL-1Ra production. Furthermore, the inhibition of STAT1-Ser(727) phosphorylation by Ly294002 did not affect STAT1 translocation, suggesting that STAT1 was not involved in sIL-1Ra gene induction. This was confirmed in monocytes that were transfected with small interfering RNA specifically targeting STAT1. Indeed, monocytes in which effective STAT1 gene knockdown was achieved were fully responsive to IFN-beta in terms of sIL-1Ra production. Taken together, the present data demonstrate that the induction of sIL-1Ra transcription and production by IFN-beta in human monocytes involved PI3K, but not STAT1 activation.