In order to investigate the role of insulin as a potential target autoantigen of cellular immunity in the prediabetic period, proliferative responses of T lymphocytes to human insulin were studied in nine islet-cell antibody (ICA)+ first-degree relatives of patients with Type I diabetes (individuals at high risk for the development of Type I diabetes, or the ‘prediabetic’group, which was never treated with insulin) and in 12 control individuals. Insulin autoantibodies were present in 6 9 (67%) of the prediabetic subjects and none of the controls. peripheral blood lymphocytes were collected on Ficoll and incubated with human insulin, control antigens, or media alone for 5–6-day and 9–10-day incubation periods. Cells were pulsed with 3 H-thymidine, harvested, and analysed in a scintillation counter. Results are expressed as stimulation index (SI= cpm with antigen/cpm without antigen), with a SI≥ 1.5 considered a positive response. Eight of nine (89%) prediabetic individuals responded positively to insulin after a 9–10-day incubation period, in contrast to four of 12 (33%) control subjects, P< 0.05. The mean proliferative response to insulin after 9–10 days' incubation was 2.1±0.4 and 1.2±0.1, for the prediabetic and control groups, respectively. The proliferative response to insulin was not directly correlated with levels of insulin autoantibodies (r=− 0.05, NS). These data suggest that most individuals at high risk for the development of Type I diabetes display a cellular immune response to insulin, and a subset of these individuals does not display a concomitant humoral immune response to insulin based on the presence or absence of insulin autoantibodies.