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In the present study, an attempt has been made to define whether cytotoxic cell subsets are activated <jats:italic>in situ<\/jats:italic> in the affected mucosa of susceptible individuals early after ingestion of a gluten\u2010containing diet. Duodenal biopsies from 11 patients with coeliac disease who repeatedly underwent endoscopic biopsy after ingestion of individually dosed amounts of gluten were used for immunohistochemistry and <jats:italic>in situ<\/jats:italic> hybridization. To identify the cell subsets expressing perforin mRNA and protein, <jats:italic>in situ<\/jats:italic> hybridization and FACS analyses were performed on cells isolated from fresh biopsies. Compared with normal mucosa, the number of intraepithelial lymphocytes containing perforin mRNA and protein increased significantly in tissue samples showing moderate or florid coeliac disease and closely paralleled the severity of morphological alteration, whereas the frequency of perforin\u2010expressing lamina propria lymphocytes increased only moderately. Cells isolated from florid biopsies that expressed perforin mRNA and protein were preferentially T\u2010cell receptor (TCR) \u03b1\u03b2 T cells. The increase in both the absolute number and the percentage of lymphocytes expressing perforin mRNA indicates <jats:italic>in situ<\/jats:italic> activation of lymphocytes within the epithelial compartment in florid coeliac disease upon ingestion of a gluten\u2010containing diet in patients predisposed to coeliac disease. Copyright \u00a9 2005 Pathological Society of Great Britain and Ireland. 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