Activation of the prostaglandin E₂ (PGE₂) EP₄ receptor, a G-protein-coupled receptor (GPCR), results in increases in intracellular cyclic AMP (cAMP) levels via stimulation of adenylate cyclase. Here we describe the in vitro pharmacological characterization of a novel EP₄ receptor antagonist, CJ-042794 (4-{(1S)-1-[({5-chloro-2-[(4-fluorophenyl)oxy]phenyl}carbonyl)amino]ethyl}benzoic acid). CJ-042794 inhibited [³H]-PGE₂ binding to the human EP₄ receptor with a mean pK_i of 8.5, a binding affinity that was at least 200-fold more selective for the human EP₄ receptor than other human EP receptor subtypes (EP₁, EP₂, and EP₃). CJ-042794 did not exhibit any remarkable binding to 65 additional proteins, including GPCRs, enzymes, and ion channels, suggesting that CJ-042794 is highly selective for the EP₄ receptor. CJ-042794 competitively inhibited PGE₂-evoked elevations of intracellular cAMP levels in HEK293 cells overexpressing human EP₄ receptor with a mean pA₂ value of 8.6. PGE₂ inhibited the lipopolysaccharide (LPS)-induced production of tumor necrosis factor α (TNFα) in human whole blood (HWB); CJ-042794 reversed the inhibitory effects of PGE₂ on LPS-induced TNFα production in a concentration-dependent manner. These results suggest that CJ-042794, a novel, potent, and selective EP₄ receptor antagonist, has excellent pharmacological properties that make it a useful tool for exploring the physiological role of EP₄ receptors.