Leukotriene B(4) (LTB(4)) is a product of eicosanoid metabolism and acts as an extremely potent chemotactic mediator for inflammation. LTB(4) exerts positive effects on the immigration and activation of leukocytes. These effects suggest an involvement of LTB(4) in several diseases: inflammatory bowel disease, psoriasis, arthritis, and asthma. LTB(4) elicits actions through interaction with one or more cell surface receptors that lead to chemotaxis and inflammation. One leukotriene B(4) receptor has been recently identified (LTB(4)-R1). In this report we describe cloning of a cDNA encoding a novel 358-amino acid receptor (LTB(4)-R2) that possesses seven membrane-spanning domains and is homologous (42%) and genetically linked to LTB(4)-R1. Expression of LTB(4)-R2 is broad but highest in liver, intestine, spleen, and kidney. In radioligand binding assays, membranes prepared from COS-7 cells transfected with LTB(4)-R2 cDNA displayed high affinity (K(d) = 0.17 nm) for [(3)H]LTB(4). Radioligand competition assays revealed high affinities of the receptor for LTB(4) and LTB(5), and 20-hydroxy-LTB(4), and intermediate affinities for 15(S)-HETE and 12-oxo-ETE. Three LTB(4) receptor antagonists, 14,15-dehydro-LTB(4), LTB(4)-3-aminopropylamide, and U-75302, had high affinity for LTB(4)-R1 but not for LTB(4)-R2. No apparent affinity binding for the receptors was detected for the CysLT1-selective antagonists montelukast and zafirlukast. LTB(4) functionally mobilized intracellular calcium and inhibited forskolin-stimulated cAMP production in 293 cells. The discovery of this new receptor should aid in further understanding the roles of LTB(4) in pathologies in these tissues and may provide a tool in identification of specific antagonists/agonists for potential therapeutic treatments.