BACKGROUND Current understanding of T cell epitopes in coeliac disease (CD) largely derives from intestinal T cell clones in vitro. T cell clones allow identification of gluten peptides that stimulate T cells but do not quantify their contribution to the overall gluten specific T cell response in individuals with CD when exposed to gluten in vivo. OBJECTIVE To determine the contribution of a putative dominant T cell epitope to the overall gliadin T cell response in HLA-DQ2 CD in vivo. METHODS HLA-DQ2+ individuals with CD and healthy controls. METHODS Subjects consumed 20 g of gluten daily for three days. Interferon gamma (IFN-gamma) ELISPOT was performed using peripheral blood mononuclear cells (PBMC) to enumerate and characterise peptide and gliadin specific T cells before and after gluten challenge. RESULTS In 50/59 CD subjects, irrespective of homo- or heterozygosity for HLA-DQ2, IFN-gamma ELISPOT responses for an optimal concentration of A-gliadin 57-73 Q-E65 were between 10 and 1500 per million PBMC, equivalent to a median 51% of the response for a "near optimal" concentration of deamidated gliadin. Whole deamidated gliadin and gliadin epitope specific T cells induced in peripheral blood expressed an intestinal homing integrin (alpha4beta7) and were HLA-DQ2 restricted. Peripheral blood T cells specific for A-gliadin 57-73 Q-E65 are rare in untreated CD but can be predictably induced two weeks after gluten exclusion. CONCLUSIONS In vivo gluten challenge is a simple safe method that allows relevant T cells to be analysed and quantified in peripheral blood by ELISPOT, and should permit comprehensive high throughput mapping of gluten T cell epitopes in large numbers of individuals with CD.