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Background: In response to infections and irritants, the bronchial epithelium releases the alarmin interleukin(IL)-33 to elicit a rapid immune response. Regulation of IL-33 following release from the cell is poorly understood. Here we characterise the lifecycle and isoforms of the IL-33 protein in vivo. Methods: 25ug of Alternaria extract was administered intranasally to BALB/c mice to release endogenous IL-33 into BAL Fluid. Human endogenous IL-33 was derived from lung explants or sputum from asthma patients. Recombinant IL-33 WT or mutant proteins were made in E. Coli . IL-33 proteins were characterised by Western Blot, mass spectrometry, ELISA, ST2-dependent functional assays and in vivo responses. Results: Mature IL-33 was rapidly oxidised in cell culture media, serum and the in vivo lung to generate an isoform with two disulphide bridges, C208–C259 and C227–C232, respectively. This resulted in an extensive conformational change with disruption of the ST2 binding site that terminated activity. A cysteine to serine mutant form of IL-33 was resistant to oxidation and was 30-fold more potent both in vitro and in vivo . Disulphide-bonded inactive IL-33 was detected following the peak of active IL-33 in lung lavage samples from animals challenged with Alternaria extract and from human lung explants. Disulphide bonded IL-33 was the most commonly detected isoform in sputum from patients with moderate-severe asthma. Conclusion: We propose oxidation as a novel mechanism for the rapid inactivation of secreted IL-33 that limits the range and duration of ST2-dependent immunological responses to airway stimuli.