Activation of T cells in the intestinal mucosa in response to gluten exposure is thought to play a key role in the pathogenesis of coeliac disease. Moreover, the response of the rectal mucosa to gluten challenge has been considered a useful predictor of gluten sensitivity in coeliac disease. In the present study, we assessed early changes in the expression of proinflammatory cytokine genes and the T cell receptor (TCR) Vbeta repertoire in the rectal mucosa of coeliac disease patients following experimental gluten challenge. Cytokine gene expression was assessed in rectal mucosal biopsies from coeliac disease subjects and controls before and after rectal gluten challenge using quantitative reverse transcription polymerase chain reaction analysis, and the TCR Vbeta repertoire was characterized using a multiprobe RNase protection assay. Marked up-regulation of expression of the C-X-C chemokine IL-8, the proinflammatory cytokine IL-1beta, and the C-C chemokine monocyte chemotactic protein-1 occurred within 24 h of rectal gluten challenge in coeliac disease subjects, but not in controls. Furthermore, these changes occurred in the absence of parallel changes in the expressed repertoire of TCR Vbeta genes in the rectal mucosa. Thus, an increased expression of proinflammatory cytokine genes precedes the expansion of antigen-specific T cell populations in the early period following experimental exposure of the rectal mucosa of coeliac disease patients to gluten. These findings provide new insights into pathways that may be involved in the activation or reactivation of coeliac disease.