The ability of mononuclear leukocytes to synthesize and secrete proteoglycans was evaluated. Using radiolabeling with H2 35SO4, it is shown that peripheral blood mononuclear cells (PBMC) and their major subpopulations (B cells, T cells, and monocytes), as well as mouse spleen cells, all secreted easily detectable proteoglycan. After 24-h labeling periods, 90% of macromolecular 35S could be detected in culture media. This material was primarily (greater than 95%) chondroitin-4-sulfate proteoglycan (CSPG). Production and secretion of CSPG could be stimulated more than 200% in PBMC and 300% in T cell populations by high concentrations of concanavalin A and phorbol 12-myristate-13-acetate; lipopolysaccharide induced a small (twofold) but reproducible increase in CSPG secretion by adherent mononuclear leukocytes. The CSPG secreted by PBMC was relatively small in size compared to chondrocyte CSPG (130,000 daltons vs. 2-4 million daltons) but possessed similar sizes of glycosaminoglycan chains and greater solubility in low ionic strength solutions. This sulfated polyanion, which was produced endogenously by leukocytes and was actively secreted, might function as a co-mediator or "second messenger" in certain immune responses.