Expression of cytokine genes in the islets of non-obese diabetic (NOD) female mice was examined. RNA samples were prepared from the islets and spleens of NOD mice at different time points at prediabetic stages during the natural disease process. Cytofluorometric analyses showed that the majority of lymphocyte infiltrates in the islets at 14 weeks of age consisted of T cells (68%). Of these, 80% of Thy1.2+ cells were CD4+ T cells. Less than 1% of in situ islet immune cells expressed a cell surface marker specific for the macrophage (Mac-1). Results of polymerase chain reaction using RNA (RT-PCR) prepared from spleens, and isolated and purified islets demonstrated that IFN-gamma message was detectable in the islets at 7 weeks of age (an early stage of insulitis). No message for this gene was detected in the spleen at any stage studied (7, 14 and 16 weeks of age). In contrast, TNF-alpha message was detected in both spleen and the islets at all stages, although the level of expression of TNF-alpha in the islets was much higher than that in the spleen. These results suggest that both cytokines are produced by in situ islet T cells, possibly activated T cells, which may be responsible for initiating or perpetuating autoimmune reactions in the islets.