A cDNA clone coding for the guinea pig leukotriene B 4 (BLT) receptor has been isolated from a lung cDNA library. The guinea pig BLT receptor has an open reading frame corresponding to 348 amino acids and shares 73% and 70% identity with human and mouse BLT receptors, respectively. Scatchard analysis of membranes prepared from guinea pig and human BLT receptor-transfected human embryonic kidney (HEK) 293 EBNA (Epstein–Bar Virus Nuclear Antigen) cells showed that both receptors displayed high affinity for leukotriene B 4 ( K d value of ∼0.4 nM) and were expressed at high levels ( B max values ranging from 9 to 12 pmol/mg protein). The rank order of potency for leukotrienes and related analogs in competition for [ 3 H ]leukotriene B 4 specific binding at the recombinant guinea pig BLT receptor is leukotriene B 4>20-OH-leukotriene B 4>12( R)-HETE ((5 Z,8 Z,10 E,12( R)14 Z)-12-hydroxyeicosatetraen-1-oic acid)>12( S)-HETE ((5 Z,8 Z,10 E,12( S)14 Z)-12-Hydroxyeicosatetraen-1-oic acid)>20-COOH-leukotriene B 4>U75302 (6-(6-(3-hydroxy-1 E,5 Z-undecadienyl)-2-pyridinyl)-1,5-hexanediol)≫leukotriene C 4=leukotriene D 4=leukotriene E 4. For the human receptor the rank order of 12( S)-HETE, 20-COOH-leukotriene B 4 and U75302 was reversed. Xenopus melanophore and HEK aequorin-based reporter gene assays were used to demonstrate that the guinea pig and human BLT receptors can couple to both the cAMP inhibitory and intracellular Ca 2+ mobilization signaling pathways. However, in the case of the aequorin-expressing HEK cells (designated AEQ17-293) transfected with either the guinea pig or human BLT receptor, expression of G α16 was required to achieve a robust Ca 2+ driven response. Leukotriene B 4 was a potent agonist in functional assays of both the guinea pig and human BLT receptors. U-75302 a leukotriene B 4 analogue which possesses both agonistic and antagonistic properties behaved as a full agonist of the guinea pig and human BLT receptors in AEQ17-293 cells and not as an antagonist. The recombinant guinea pig BLT receptor will permit the comparison of the intrinsic potencies of leukotriene B 4 receptor antagonists used in guinea pig in vivo models of allergic and inflammatory disorders.