Peripheral blood lymphocytes were obtained from 65 individuals: 34 nondiabetic patients with islet cell autoantibodies (ICA) (prediabetic phase), 9 patients with newly diagnosed insulin-dependent diabetes mellitus (IDDM), 6 ICA-negative siblings or offsprings of IDDM patients, and 16 ICA-negative controls. The presence of lymphocyte abnormalities and/or activation was examined with dual fluorescence flow cytometry. The percentages of B cells, total T-lymphocyte, and helper T-lymphocyte (Th) and cytotoxic/suppressor T-lymphocyte (Tc/s) subsets and their ratio were not significantly different among the patient groups. No increased expression of interleukin 2 receptor on T-lymphocyte was found in newly diagnosed IDDM or prediabetic individuals. Sixteen of 49 patients had significantly increased number of T-lymphocyte expressing HLA-DR. A significant increase in the number of both Th and Tc/s subsets expressing HLA-DR was found in only 3 of 16 patients. This increase was unrelated to the patients's relative ICA titer or HLA-DR phenotype. On the other hand, the relative density of the DR antigen (RAD-DR) was significantly increased on both Th (886 +/- 120) and Tc/s (1250 +/- 273) in 13 of 38 patients compared with control patients (Th 484 +/- 129 and Tc/s 460 +/- 166). The RAD-DR on Tc/s correlated with the relative ICA titer and was greatest on DR3/4-phenotyped T-lymphocytes. In addition, significantly increased RAD-DR was found in noncontrol patients with impaired insulin release responses at 1 and 3 min to intravenous glucose.(ABSTRACT TRUNCATED AT 250 WORDS)