Using en face double immunostaining coupled with electron microscopy, we studied the temporal and spatial distribution of T lymphocytes and macrophages during the development of atherosclerosis in a diet-induced rat model fed an atherogenic diet for 2-40 weeks. T lymphocytes and macrophages adhered to the aortic surface by 2 weeks on the diet, with subsequent migration under the endothelium, and formed a fatty streak-like lesion. Analysis of the cellular components revealed that infiltration of T lymphocytes was most prominent in the incipient phase of lesion formation accounting for 60%, 29% and 34% of mononuclear cells appearing in 2-week lesions of the superior thoracic, inferior thoracic and abdominal segments of the aorta, respectively. After the incipient phase, the relative number of T lymphocytes in the three segments of the aorta showed a slow decline; the proportion of T lymphocytes to macrophages was approximately 1:3 to 1:4 in 10- to 20-week lesions. An overall view of the lesional cells often demonstrated direct cellular contact between T lymphocytes and macrophages. Further, OX6/ED1 double immunostaining demonstrated that Ia antigen was expressed on most macrophages. In later stages, breakdown of foamy macrophages occurred, and the extracellular accumulation of lipids and cell debris became prominent. The results demonstrated that in the diet-induced rat model, together with macrophages, large numbers of T lymphocytes participated in all stages of aortic lesions, initially adhering to the surface at prelesional stages and later as the principal component of the atherosclerotic lesion. It is possible that the method described here will provide a good tool for examining the role of T lymphocytes in atherogenesis.