We studied the origin of the neutrophil-activating peptide NAP-2, a presumed 70 amino acid cleavage product of platelet basic protein (PBP) and connective tissue-activating peptide III (CTAP-III). Purified human blood monocytes or lymphocytes were cultured with or without stimuli (LPS or PHA) in the presence or absence of platelet-release supernatant, and the formation of NAP-2 and other neutrophil-activating peptides was monitored. NAP-2 was generated whenever monocytes and platelet release supernatant were present. When a monocyte stimulus was added, NAF/NAP-1 was also formed, and in the presence of LPS a third, less potent neutrophil-stimulating fraction, consisting of NAP-2 variants with 73, 74, and 75 residues, also appeared. Monocytes alone did not yield NAP-2 and no neutrophil-activating peptide was generated by lymphocytes. The monocyte-conditioned medium was found to cleave purified CTAP-III into NAP-2 through proteinases that were highly sensitive to PMSF, moderately sensitive to leupeptin and insensitive to EDTA.