Neutrophil peptide, also called defensin, is an antimicrobial peptide localized in azurophil granules in neutrophils. Using a synthetic human neutrophil peptide-1 (HNP-1), we prepared an antiserum against the peptide and established a highly sensitive radioimmunoassay (RIA). This RIA equally recognized HNP-1, -2, and -3 on a molar basis. Analyses of leukocyte and blood extracts obtained from normal individuals and patients with bacterial infection by reverse-phase high-performance liquid chromatography coupled with the RIA showed that the antiserum specifically detected HNP molecules. Concentrations of HNP 1-3 in total in normal human blood and plasma are 13.2 ± 1.4 (mean ± SEM) ng/μ1 and 403.8 ± 38.9 pg/μ1, respectively. The values increased in infection and returned to within the normal levels after the recovery of the disorder. Granulocyte colony-stimulating factor (G-CSF) increased the plasma and blood concentrations of HNP 1-3. The RIA established here provides a useful tool to elucidate the pathophysiological significance of HNP and to evaluate the neutrophil functions in infection and inflammation.