Lysophosphatidic acid (LPA) exerts a variety of biological responses through specific receptors: three subtypes of the EDG-family receptors, LPA 1 , LPA 2 , and LPA 3 (formerly known as EDG-2, EDG-4, and EDG-7, respectively), and LPA 4 /GPR23, structurally distinct from the EDG-family receptors, have so far been identified. In the present study, we characterized the action mechanisms of 3-(4-[4-([1-(2-chlorophenyl)ethoxy]carbonyl amino)-3-methyl-5-isoxazolyl] benzylsulfanyl) propanoic acid (Ki16425) on the EDG-family LPA receptors. Ki16425 inhibited several responses specific to LPA, depending on the cell types, without any appreciable effect on the responses to other related lipid receptor agonists, including sphingosine 1-phosphate. With the cells overexpressing LPA 1 , LPA 2 , or LPA 3 , we examined the selectivity and mode of inhibition by Ki16425 against the LPA-induced actions and compared them with those of dioctyl glycerol pyrophosphate (DGPP 8:0), a recently identified antagonist for LPA receptors. Ki16425 inhibited the LPA-induced response in the decreasing order of LPA 1 Ն LPA 3 Ͼ Ͼ LPA 2 , whereas DGPP 8:0 preferentially inhibited the LPA 3 -induced actions. Ki16425 inhibited LPA-induced guanosine 5Ј-O-(3-thio)triphosphate binding as well as LPA receptor binding to membrane fractions with a same pharmacological specificity as in intact cells. The difference in the inhibition profile of Ki16425 and DGPP 8:0 was exploited for the evaluation of receptor subtypes involved in responses to LPA in A431 cells. Finally, Ki16425 also inhibited LPA-induced longterm responses, including DNA synthesis and cell migration. In conclusion, Ki16425 selectively inhibits LPA receptor-mediated actions, especially through LPA 1 and LPA 3 ; therefore, it may be useful in evaluating the role of LPA and its receptor subtypes involved in biological actions.Lysophosphatidic acid (LPA) has been shown to elicit diverse biological actions, including Ca 2ϩ mobilization, change in cAMP accumulation, change in cell shape and motility in association with actin rearrangement, and proliferation in a variety of cell types (Moolenaar, 1999;Contos et al., 2000;Ye et al., 2002). Extracellular LPA has also been shown to be involved in certain diseases, such as atherosclerosis and cancer (Xu et al., 1995(Xu et al., , 2001Siess et al., 1999;Maschberger et al., 2000). LPA was first thought to be released from activated platelets; however, a major part of extracellular LPA has been shown to be produced from lysophosphatidylcholine by lysophospholipase D, which was previously called autotaxin (Sano et al., 2002;Tokumura et al., 2002;Umezu-Goto et al., 2002). The concentration of plasma LPA is about 100 nM, and its serum concentration can be as high as 5 M (Sano et al., 2002). LPA increases low-density lipoprotein during its oxidation, activates endothelial cells (Siess et al., This work was supported in part by a research grant grants-in-aid for scientific research from the Japan Society for the Promotion of Science and by research gr...