CD40–CD40L receptor–ligand interaction plays a central role in antigen presentation, immunological reactions, and in T-cell and macrophage activation. Since all these mechanisms are important for the pathogenesis of atherosclerosis, we studied the expression profile of CD40–CD40L in different types of human atherosclerotic lesions using double immunostaining techniques with cell type-specific antibodies. Normal human intima did not contain CD40 or CD40L immunoreactivity. From type-II lesions (fatty streaks) to advanced type-VI lesions (complicated plaques), colocalization of CD40 and CD40L was observed in T cells (CD3⁺ cells), macrophages (CD68⁺ cells), and smooth muscle cells (HHF35⁺ cells). No correlation was found between the lesion type and CD40–CD40L expression. Positive lesions had dense infiltrations of macrophages and macrophage-derived foam cells together with T cells. The most intensive immunoreactivity for the CD40 receptor and its ligand CD40L was found in macrophage- and T-cell-rich pockets, where both cell types were in close contact with each other. The majority of macrophages, and especially those of macrophage-derived foam cells, were positive for both CD40 and CD40L. A small subset of the lesion macrophage population (10–20%) consisted of cells positive only for either CD40 or CD40L, suggesting the presence of a subpopulation of macrophages more active in inflammatory processes than in lipid uptake. Intimal smooth muscle cells in and around the macrophage-rich areas as well as some of the medial smooth muscle cells near the lesions stained positive for CD40 and CD40L. Moderate to faint expression of these proteins was also found in endothelium. In addition, CD40–CD40L immunoreactivity colocalized with epitopes characteristic of oxidized low-density lipoprotein, scavenger receptor class A, and CD16 (FcγRIII), thus suggesting the involvement of CD40–CD40L and these pathogenetic mediators in foam cell formation, progression of atherosclerotic lesions, and differentiation of immunologically active subsets of macrophages. These results support the hypothesis that CD40–CD40L interaction is involved in atherogenesis and that it might provide a target for future therapeutic interventions.