CD40 is a prominent B cell Ag also found on certain epithelial cells and on carcinomas. In this report, we analyzed CD40 distribution in the human thymus. CD40 was not found on the majority of CD45-positive thymocytes, but was present in a CD45-negative stromal cell population. Immunohistology showed CD40 expression on cortical thymic epithelial cells (TEC), medullary TEC, thymic interdigitating cells, and thymic B cells. CD40 was not found on thymocytes, endothelial cells, or on the fibroblasts of the septae. Expression of CD40 was specifically maintained on cultured TEC and not found on cultured thymic fibroblasts. IL-1 alpha, TNF-alpha, IFN-gamma, but not IL-4, significantly up-regulated the membrane expression of CD40 on cultured TEC. The regulation of CD40 was similar to that of ICAM-1, and contrasted with that of MHC class II Ag, which could only be induced by IFN-gamma but not by IL-1, TNF, or IL-4. Northern blot analysis showed the presence of a 1.4-kb mRNA transcript for CD40 in TEC, which was up-regulated by IL-1 and IFN-gamma. Cross-linking of CD40 at the surface of human TEC in the absence of IL-1 stimulation failed to induce cytokine secretion. Triggering of TEC with anti-CD40 mAb in conjunction with IFN-gamma and IL-1 stimulation increased granulocyte-macrophage CSF secretion in a dose-dependent manner. The effect was visible as early as 24 h after triggering, occurred in the absence of cellular proliferation, and was specific for CD40 since triggering of other TEC membrane Ag such as ICAM-1 or MHC class I molecules had no effect to increase cytokine production in TEC. These data demonstrate that CD40 is expressed and is a functional molecule at the surface of the epithelial cells of the thymus.