We have previously shown that long-term treatment of airway smooth muscle (ASM) cells with a combination of TNF-alpha and IFN-gamma impaired steroid anti-inflammatory action through the up-regulation of glucocorticoid receptor beta isoform (GRbeta) (Mol Pharmacol 2006;69:588-596). We here found that steroid actions could also be suppressed by short-term exposure of ASM cells to TNF-alpha and IFN-gamma (6 h) as shown by the abrogated glucocorticoid responsive element (GRE)-dependent gene transcription; surprisingly, neither GRalpha nuclear translocation nor GRbeta expression was affected by cytokine mixture. The earlier induction of CD38, a molecule recently involved in asthma, seen with TNF-alpha and IFN-gamma 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-alpha-induced IRF-1 expression, IRF-1 DNA binding, and transactivation activities was completely lost in cells exposed to TNF-alpha and IFN-gamma in 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 GRbeta-independent mechanism mediating steroid dysfunction induced by pro-asthmatic cytokines, in part via the suppression of GRalpha activities.