Neutrophil-activating peptide ENA-78 is a novel chemotactic cytokine isolated from a human type II pulmonary epithelial cell line. It is a member of the chemokine family of proinflammatory polypeptides and exhibits structural homology to interleukin-8 (IL-8) and GROalpha. The immunohistochemical identification of ENA-78 in pulmonary alveolar leukocytes of bovine pneumonic lungs supports a role for ENA-78 in the pathogenesis of pulmonary inflammation. Although ENA-78 is able to stimulate polymorphonuclear neutrophils (PMN), neither its binding specificities nor its expression in human pulmonary disease states have been determined. 125I-labeled ENA-78 binds with high affinity to human PMN. Its actions on PMN appear to be mediated by the IL-8 type B receptor, to which it binds with a K(d) of 2.2 nM. Human IL-8, GROalpha, and murine KC compete with high affinity for 125I-ENA-78 binding to the human IL-8 type B receptor. In contrast, 125I-ENA-78 does not bind to the IL-8 type A receptor nor does it compete significantly for 125I-IL-8 binding to this same receptor. ENA-78 is a potent upregulator of Mac-1 cell surface expression. In addition, ENA-78 mRNA is detected in cystic fibrosis lung but is not detected in normal donor lung. Thus, ENA-78 mRNA levels appear to be increased in human pulmonary inflammation and its stimulatory activities on PMN appear to be a function mediated primarily by the IL-8 type B receptor.