In order to identify any dominating subset of activated T cells in the celiac lesion, we examined CD3+, CD4+, CD8+ and T cell receptor (TcR) gamma/delta+ lymphocytes in jejunal cryosections from 25 patients with celiac disease and 10 controls by three-color immunofluorescence staining for expression of the nuclear proliferation marker detected by monoclonal antibody (mAb) Ki-67 and the p55 alpha chain of interleukin-2 receptor (CD25). mAb Ki-67+ intraepithelial lymphocytes (IEL) were exclusively observed in celiac patients. The median proportion of CD3+ IEL positive for Ki-67 increased from nil in controls to 4.5% in partly treated (range 0-19.0%; n = 10; p = < 0.05) and 12.8% in untreated celiac disease (range 4.0-30.7%; n = 15; p < 0.005). Only 1.5% of CD3+ subepithelial T cells expressed the Ki-67 marker in celiac disease (range 0-9.5%). Two- and three-color staining combining mAb to CD3 and Ki-67 with mAb to CD4, CD8 or TcR delta showed that both TcR alpha/beta+ CD8+ and TcR gamma/delta+ (but not CD4+) mucosal T cells proliferated in the epithelium. By contrast, CD25 were almost exclusively expressed on CD4+ T cells in the lamina propria. The percentage of CD25+ T cells increased significantly from 1.7% in controls (range 0-2.9%) to 7.5% in partly treated (range 0.8-17.8%, p < 0.002), and to 14.65% in untreated celiac disease (range 3.9-21%, p < 0.002). These results suggest that gluten ingestion in celiac disease induces proliferative activation of TcR alpha/beta+ CD8+ and TcR gamma/delta+ IEL but non-proliferative activation (lymphokine production?) of lamina propria CD4+ T cells.