We have described a procedure for isolating thymic macrophages and have evaluated their activity in stimulating thymocyte maturation in vitro. The culturing of gradient-purified immature thymocytes on thymic macrophages leads to an increased expression of H-2 antigens and decreased lytic sensitivity with anti-TL and C. The macrophage-stimulated thymocyte also acquires the ability to respond in the MLR. We propose that the macrophage may regulate one stage of thymic differentiation in vivo.