Lipoxin A4 (LXA4) interacts with high-affinity receptors in human neutrophils and differentiated HL-60 cells. Recently, we characterized a myeloid-derived cDNA that encodes a LXA4 high-affinity receptor (LXA4R) [Fiore, S., Maddox, J. F., Perez, H. D., and Serhan, C. N. (1994) J. Exp. Med. 180, 253-260] denoted earlier as a related N-formyl peptide receptor (RFP). To examine the selectivity of this receptor we tested its preference for specific binding of 3H-LXA4 versus 3H-N-formylmethionyl-leucyl-phenylalanine (3H-FMLP). When receptor-transfected Chinese hamster ovary cells were exposed to either 3H-LXA4 or 3H-FMLP, the receptor affinity for LXA4 exceeded by 1000-fold that of FMLP (6.1 nM vs 5 microM). Upon differentiation, HL-60 cells acquire high-affinity binding sites and respond to both LXA4 and FMLP. Northern blot analysis of differentiated HL-60 cells using an RFP probe showed a characteristic band at 2.1 kb. Differentiated HL-60 cells exposed to an RFP antisense oligonucleotide selectively lost 3H-LXA4 binding as well as LXA4-stimulated lipid remodeling that paralleled the loss of mRNA for LXA4R. In contrast, the specific mRNA for the FMLP receptor, 3H-FMLP specific binding, and FMLP-induced phospholipase D activity were still observed. Treatment of human neutrophils with antisera raised against a peptide in the LXA4R third extracellular domain also resulted in selective abrogation of 3H-LXA4 specific binding with polymorphonuclear leukocytes (PMN) without blocking 3H-FMLP binding. FMLP-stimulated CD11b upregulation as well as homotypic aggregation of PMN was inhibited by LXA4 (which at 10(-9) M gave approximately 1 log unit shift to the right in the FMLP dose-response curve). The addition of LXA4R antisera did not alter FMLP-induced responses in PMN but completely blocked LXA4 actions. These results indicate that altering the expression of the LXA4R protein by blockage of transcriptional mechanisms or hindrance of the LXA4R extracellular domains leads to loss of LXA4 specific binding and blockage of LXA4 signaling. Moreover, they indicate that in myeloid cells LXA4-LXA4R interactions are dissociable from those of FMLP and that LXA4 regulates CD11/18 on the PMN surface.