Mast cells are central in the development of several allergic diseases and contain a number of pre-formed mediators. -tryptase, the most abundant mast cell product, is increasingly recognized as a key inflammatory mediator as it causes the release of cytokines, particularly the chemokine IL-8, from both inflammatory and structural cells. The molecular mechanisms, however, remain largely unknown. In this study we sought to investigate whether -tryptase could induce IL-8 expression in human airway smooth muscle (ASM) cells and to explore the molecular mechanisms involved. We found that purified human -tryptase stimulated IL-8 production time- and concentration-dependently, which was inhibited by protease inhibitors and mimicked by recombinant human -tryptase, but not by the protease-activated receptor-2 (PAR-2) agonist SLIGKV-NH₂, consistent with the low level expression of PAR-2 protein in these cells. -tryptase also up-regulated IL-8 mRNA expression, as analyzed by RT-PCR and real-time PCR, which was abolished by the transcription inhibitor actinomycin D. Reporter gene assay showed that -tryptase-induced IL-8 transcription was mediated by the transcription factors AP-1, C/EBP and NF-B, and chromatin immunoprecipitation assay demonstrated that -tryptase induced in vivo binding of these transcription factors to the IL-8 gene promoter. Furthermore, -tryptase stabilized IL-8 mRNA, suggesting additional post-transcriptional regulation. Collectively these findings show that -tryptase upregulates IL-8 expression in ASM cells through a PAR-2-independent proteolytic mechanism and coordinated transcriptional and post-transcriptional regulation, which may be of particular importance in understanding the role and the mechanisms of action of -tryptase in regulating chemokine expression in mast cell-related disorders.