Interferon-beta (IFN-beta) is a critical antiviral cytokine and is essential for innate and acquired immune responses to pathogens. Treatment with polyinosinic:polycytidylic acid (poly(I:C)) induces transient accumulation of IFN-beta mRNA, which involves an increase and a decrease of IFN-beta mRNA. This phenomenon has been extensively analyzed as a model for understanding the mechanisms of transient gene induction in response to external stimuli. Using a new RNA metabolic labeling method with ethynyluridine to directly measure de novo RNA synthesis and RNA stability, we reassessed both de novo synthesis and degradation of IFN-beta mRNA. We found that transcriptional activity is maintained after the maximum accumulation of IFN-beta mRNA following poly(I:C) treatment on immortalized human bronchial epithelial cells. We also observed an unexpected change in the stability of IFN-beta mRNA before and after the maximum accumulation. The results indicate that this method of RNA metabolic labeling provides a general approach for the simultaneous analysis of transcriptional activity and mRNA stability coupled with transcriptional timing. (C) 2012 Elsevier Inc. All rights reserved.