Diverse conditions for stimulating human mononuclear cells to release thymocyte costimulatory factors were tested for their contribution to the generation of supernatants containing high titers of these monokines. Activity titers increased with LPS concentration, reaching a plateau between 1 and 10 micrograms/ml. Indomethacin did not modify the monokine release, but the assay for thymocyte costimulatory activity was substantially affected by inhibitory substances produced by the monocytes in the absence of indomethacin. The use of nylon wool columns to trap the cells was shown to be effective in raising cellular densities without decreasing activity titers. As a result, the yield per cell could be maintained even in the absence of serum, an important step toward the goal of purifying bioactive peptides from crude broths.