We have previously shown that long-term treatment of airway smooth muscle (ASM) cells with a combination of TNF and IFN impaired steroid anti-inflammatory action through the upregulation of glucocorticoid receptor beta isoform (GR) (Mol Pharmacol. 2006 Feb;69(2):588-96). We here found that steroid actions could also be suppressed by short-term exposure of ASM cells to TNF and IFN (6 h) as evidenced by the abrogated glucocorticoid responsive element (GRE)-dependent gene transcription; surprisingly neither GR nuclear translocation nor GR expression was affected by cytokine mixture. The earlier induction of CD38, a molecule recently involved in asthma, seen with TNF and IFN combination, but not with cytokine alone, was also completely insensitive to steroid pretreatment. Chromatin-immunoprecipitation (IP) and siRNA strategies revealed not only increased binding of Interferon Regulatory Factor 1 (IRF-1) transcription factor to CD38 promoter but also its implication in regulating CD38 gene transcription. Interestingly, the capacity of fluticasone to completely inhibit TNF-induced IRF-1 expression, IRF-1 DNA binding, and transactivation activities, was completely lost in cells exposed to TNF and IFN combination. This early steroid dysfunction seen with cytokine combination could be reproduced by enhancing IRF-1 cellular levels using constitutively active IRF-1 which dose-dependently inhibited GRE-dependent gene transcription. Consistently, reducing IRF-1 cellular levels using siRNA approach significantly restored steroid transactivation activities. Collectively, our findings demonstrate for the first time that IRF-1 is a novel alternative GR-independent mechanism mediating steroid dysfunction induced by pro-asthmatic cytokines, in part via the suppression of GR activities.