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Coeliac disease (CD) is a T-cell mediated immunological disease of the small intestine which is triggered in susceptible individuals by ingestion of gluten. The pathogenic mechanism of coeliac disease, and the role that alpha-gliadin specific T cells play in mucosal lesions and their involvement in peripheral blood is not yet explained at all. Previous studies have reported proliferative response to alpha-gliadin measured with the classic assay of 3HTdR incorporation. We analysed the activation antigen CD69 on T cells from CD patients and normal individuals following stimulation with alpha-gliadin and different antigens (tetanus toxoid, peptides unrelated to gliadin and PHA). CD69 coexpression with T cell CD3+ and proliferation marker Ki67 was evaluated with time. CD69 coexpression with T cell CD3+, CD4+ and CD8+ was also evaluated. It was found that peripheral blood mononuclear cells (PBMC) of coeliac patients increased their percentage of CD69 positive T cells when stimulated with alpha-gliadin, in comparison with cells from controls. Significant T cell activation was found only in subjects not treated with the gluten free diet; a positive response was found also in two coeliac patients with selective IgA deficiency, anti-endomisium negative, without circulating IgA anti alpha-gliadin or anti-tissue transglutaminase antibodies. The CD69 expression after stimulation was compared with the standard method of 3HTdR incorporation. Our data show that CD69 expression is useful to asses a specific T cell response to alpha-gliadin in coeliac disease. in a very short time. Moreover, the method allows to investigate T cell response at the lymphocyte subsets level, which represents a useful tool in the diagnosis of coeliac disease.