Cyclosporin A (CsA) is a fungus-derived cyclic undecapeptide with potent immunosuppressive activity. Its analog, cyclosporin H (CsH), lacks immunosuppressive function but can act as an antagonist for the human formyl peptide receptor (FPR). More recent studies have shown that CsA also inhibits fMLF-induced degranulation in differentiated HL-60 promyelocytic leukemia cells. However, it is unclear whether CsA interferes with ligand-receptor interaction, G protein activation, or other downstream signaling events. In this study we used human neutrophils, differentiated HL-60 cells, and rat basophilic leukemia (RBL)-2H3 cells expressing human FPR (RBL-FPR) to identify the action site of CsA. In functional assays, CsA inhibited fMLF-stimulated degranulation, chemotaxis, calcium mobilization, and phosphorylation of the MAPKs ERK 1/2 and the serine/threonine protein kinase Akt. CsA also blocked Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm)-induced functions in RBL-FPR cells. Concentrations for half-maximal inhibition with CsA are generally 6- to 50-fold higher than that of CsH. CsA was compared with another immunosuppressant, ascomycin, relative to the inhibitory effects on FPR-mediated chemotaxis, calcium mobilization, and degranulation. In these experiments, ascomycin produced no inhibitory effects at low micromolar concentrations (1-4 microM), whereas the inhibitory effects of CsA were prominent at comparable concentrations. Finally, CsA dose-dependently inhibited the uptake of fNle-Leu-Phe-Nle-Tyr-Lys-fluoresceine and [3H]fMLF or [125I]WKYMVm binding to FPR. However, CsA and CsH did not show any obvious inhibitory effect on FPR-like 1-mediated cellular functions. These results demonstrate that CsA is a selective antagonist of FPR and that its inhibition of fMLF-stimulated leukocyte activation is at the level of cognate ligand binding.