Dendritic cells (DC), with their potent antigen-presenting and accessory activities, are involved in the stimulation of naive T cells. To examine the biological functions of DC, we developed a method for generating large numbers of murine splenic DC. First, DC were propagated in vivo by using a granulocyte-macrophage colony-stimulating factor-secreting tumor as a continuous in vivo source of the cytokine. The DC enriched in the spleen, especially in the white pulps, were purified after an overnight culture. We could reproducibly obtain 6 to 10 X 10(6) splenic DC per mouse. These DC were morphologically similar to interdigitating cells, expressed high levels of MHC class II and costimulatory molecules, and were highly allo-stimulatory in mixed lymphocyte reactions. Further analysis on T cell stimulation activity revealed that the DC had strong costimulatory activity on human T cells. Activated B cells, which express both B7-1 and B7-2, had little T cell costimulatory activity under the same assay conditions. A human histiocytic leukemia cell line, U937, that showed only weak costimulatory activity by itself, worked synergistically with DC and further intensified the T cell stimulation by DC. These findings suggest the presence of a T cell costimulation mechanism in DC, which is activated synergistically by monocytes or macrophages, and deserves further study.