Culture medium conditioned by stimulating human mononuclear leucocytes (MNL) with killed Staphylococcus aureus (Scm) was found to contain a substantial amount of tumour necrosis factor-alpha (TNF-alpha) but no detectable tumour necrosis factor-beta (TNF-beta). Culture medium conditioned by MNL in the absence of bacteria contained no TNF-alpha activity. When Scm was fractionated by high-performance liquid chromatography (HPLC) on Bio-Sil TSK 250, TNF-alpha co-eluted with neutrophil-stimulating activity measured by chemiluminescence. Similarly, the ability of neutrophils to kill opsonized S. aureus was enhanced in fractions that contained this neutrophil-stimulating activity. The stimulating activity could be almost completely removed by pretreatment of the Scm with a TNF-alpha-specific monoclonal antibody (mAb). The ability of neutrophils to kill S. aureus in response to Scm was also substantially reduced by mAb to TNF-alpha. These results demonstrate that bacterial interaction with MNL leads to the release of neutrophil-stimulating activity that consists predominantly of TNF-alpha.