The differentiation of macrophages present in diffuse intimal thickening, fatty streaks, and atheromatous plaques, was analyzed with immunohistochemical methods, using segments of aorta, coronary, and carotid arteries obtained at autopsy. Various differentiation antigens were studied with the monoclonal antibodies anti-HLA-DR, EBM-11, Leu M3, OKM1, and OKM5. Adjacent sections were stained for lipids (oil red O) and lysosomal activity (acid phosphatase). Almost all macrophages identified with the pan-macrophage antibody EBM-11, also stained with the anti-HLA-DR antibody. Diffuse intimal thickening showed a predominance of Leu M3+ cells; fatty streaks also showed OKM1+ and OKM5+ macrophages. Classical atheromatous plaques showed a gradual shift in phenotypic expression towards the center of the lesion. Cells in the superficial layers were positive only with Leu M3, deeper localized cells showed double expression of Leu M3 and OKM1 or double expression of OKM1 and OKM5. Cells that were localized adjacent to the atheromatous debris stained only with OKM5. The phenotypic changes occurred in parallel with an increase in both fat uptake and lysosomal activity of the macrophages. This shift in phenotypic expression suggests a process of differentiation and maturation of the macrophages involved. The results indicate that macrophages within the arterial intima are activated and mature towards cells that express receptors for adhesion proteins and complement during the development of atherosclerotic plaques. This may imply that the macrophages involved in lipid metabolism also have a potential to act as effector cells in a chronic inflammatory process, and thus, may contribute to the progression of an atherosclerotic plaque. Functional studies of macrophage subpopulations are needed to verify this hypothesis.