The rise in cytosolic free Ca2+, shape change, superoxide formation, and granule exocytosis induced in human neutrophils by N‐formyl‐Met‐Leu‐Phe (fMLP) and by a newly discovered activating peptide, neutrophil‐activating factor, termed NAF, were compared. NAF was effective in the concentration range of 0.1‐10 nM and was 10‐to 100‐fold more potent than fMLP. In qualitative terms, the single responses to either stimulus were remarkably similar: they showed virtually identical onset and initial kinetics, and were all inhibited by pretreatment of the neutrophils withBordetella pertussistoxin. In addition, the respiratory burst elicited by either stimulus was inhibited by 17‐hydroxywortmannin and staurosporine. Two conclusions are drawn from these results:1)neutrophil activation by NAF (as by fMLP) is dependent on a GTP‐binding protein and on protein kinase C;2) a similar, or even identical, mechanism of signal transduction must be assumed on stimulation of human neutrophils with NAF, fMLP, and other chemotactic agonists. Human monocytes, lymphocytes, and platelets did not show cytosolic free Ca2+changes when exposed to NAF, which suggests that NAF is selective for the neutrophils.— Thelen, M.; Peveri, P.; Kernen, P.;vonTscharner, V; Walz, A.; Baggiolini, M. Mechanisms of neutrophil activation by NAF, a novel monocyte‐derived peptide agonist.FASEB J.2: 2702‐2706; 1988.