OBJECTIVE: The analysis of intraepithelial lymphocytes (IELs) by flow cytometry of duodenal biopsies-the 'IEL' lymphogram-has been proposed as a diagnostic test for coeliac disease. However, its clinical applicability has been limited due to variability in methods and definitions. This study set out to define useful parameters for the application of the IEL lymphogram to the diagnosis of coeliac disease. DESIGN: Flow cytometry was performed on 117 sets of duodenal biopsies in 107 adult patients with active coeliac disease, long-term coeliac disease on a gluten free diet and a control group. The initial 95 samples were used for hypothesis generation for the subsequent samples comprising 12 patients with coeliac disease and 10 controls. RESULTS: Rather than using single linear cut-offs for CD3 and T-cell receptor γδ (TCRγδ)+ve IELs, a discriminant function was identified as %CD3+ve IELs+2x(%TCRγδ+IELs)>100. This differentiated coeliac disease from control biopsies in the hypothesis generating group. These results were replicated in the validation group and found to be independent of histology in patients on long-term gluten free diet up to 12 years (combined sensitivity, 98.5%; specificity, 97.7%). CONCLUSIONS: Flow cytometric analysis of IELs is a highly sensitive and specific adjunct to serology and histological examination for the diagnosis of coeliac disease, even in individuals with coeliac disease following a gluten free diet who exhibit normal duodenal histology.