Culture medium conditioned by human mononuclear leukocytes (MNL) stimulated with formalin fixed heat-killed Staphylococcus aureus induces a small respiratory burst in human neutrophils, and dramatically increases the response of neutrophils to stimuli such as N-formyl-L-methionyl-L-leucyl-L-phenylalanine. The data presented show that the activity is not unique to Staphylococcus aureus. Similar neutrophil modulating activities were produced by medium conditioned by MNL cultured in the presence of Streptococcus pneumonia, and Group B streptococcus. The activity was relatively resistant to heating; significant reduction of activity was observed only when 80 degrees C was reached. Neutrophil stimulating activity production by stimulated MNL was dependent on protein and RNA synthesis and the activity appeared to be released by the non-adherent fraction of the MNL, suggesting that it is not of macrophage origin. The activity was not sensitive to soya bean trypsin inhibitor, but was sensitive to trypsin and was not removed when stimulated conditioned medium was depleted of immunoglobulin and albumin by affinity chromatography. Purification by gel filtration on Sephadex G-100 and high-performance liquid chromatography with Bio-Sil TSK250 columns showed that the major activity had an apparent molecular weight of 35,000-43,000 under conditions in which ionic interactions and association with albumin were reduced; by using polyethylene glycol or high salt (0.46 M Na+) in the elution buffer.