Endothelial-monocyte-activating polypeptide II (EMAP II) is a novel mediator isolated from conditioned medium of methylcholanthrene A-induced tumor cells which modulates properties of endothelial cells, mononuclear phagocytes (MPs), and polymorphonuclear leukocytes (PMNs) in vitro and induces an acute inflammatory response in vivo. A synthetic peptide comprising 15 residues from the N-terminal region (residues 6-20) was shown to induce directional migration of MPs and PMNs, with half-maximal effect at approximately 200-250 pM, whereas a peptide from the C terminus of EMAP II, as well as other irrelevant peptides, were without effect. Modulation of cellular phenotype by EMAP II-derived peptide was suggested by peptide-induced elevation of cytosolic free calcium concentration in fura-2-loaded MPs and PMNs and by stimulation of peroxidase release in PMNs. Consistent with these in vitro data, EMAP II-derived N-terminal peptide-albumin conjugates injected into the mouse footpad elicited inflammatory cell tissue infiltration, whereas albumin alone or EMAP II-derived C-terminal peptide conjugated to albumin incited little response. Binding of 125I-labeled EMAP II-derived peptide (residues 12-20) to MPs was saturable (Kd approximately 200 pM) and was blocked in a dose-dependent manner by the addition of intact EMAP II and unlabeled EMAP II-derived peptides (residues 6-20 and 12-20), whereas interleukin 1, tumor necrosis factor, formyl-methionyl-leucinyl-phenylalanine, or irrelevant peptides were without effect. Cross-linking of 125I-EMAP II-derived peptide (residues 12-20) by disuccinimidyl suberate to human MPs demonstrated a band, approximately 73 kDa, on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 125I-EMAP II-derived peptide also demonstrated specific binding to human PMNs and murine RAW cells. These data indicate that the N-terminal region of EMAP II defines a biologically active locus of the molecule which interacts with target cells via a potentially novel cellular receptor.