Lysophosphatidic acid (LPA), via interaction with its G-protein coupled receptors, is involved in various pathological conditions. Extracellular LPA is mainly produced by the enzyme autotaxin (ATX). Using fibroblast-like synoviocytes (FLS) isolated from synovial tissues of patients with rheumatoid arthritis (RA), we studied the expression profile of LPA receptors, LPA-induced cell migration, and interleukin (IL)-8 and IL-6 production. We report that FLS express LPA receptors LPA(1-3). Moreover, exogenously applied LPA induces FLS migration and secretion of IL-8/IL-6, whereas the LPA(3) agonist l-sn-1-O-oleoyl-2-methyl-glyceryl-3-phosphothionate (2S-OMPT) stimulates cytokine synthesis but not cell motility. The LPA-induced FLS motility and cytokine production are suppressed by LPA(1/3) receptor antagonists diacylglycerol pyrophosphate and (S)-phosphoric acid mono-(2-octadec-9-enoylamino-3-[4-(pyridine-2-ylmethoxy)-phenyl]-propyl) ester (VPC32183). Signal transduction through p42/44 mitogen-activated protein kinase (MAPK), p38 MAPK, and Rho kinase is involved in LPA-mediated cytokine secretion, whereas LPA-induced cell motility requires p38 MAPK and Rho kinase but not p42/44 MAPK. Treatment of FLS with tumor necrosis factor-alpha (TNF-alpha) increases LPA(3) mRNA expression and correlates with enhanced LPA- or OMPT-induced cytokine production. LPA-mediated superproduction of cytokines by TNF-alpha-primed FLS is abolished by LPA(1/3) receptor antagonists. We also report the presence of ATX in synovial fluid of patients with RA. LPA(1/3) receptor antagonists and ATX inhibitors reduce the synovial fluid-induced cell motility. Together the data suggest that LPA(1) and LPA(3) may contribute to the pathogenesis of RA through the modulation of FLS migration and cytokine production. The above results provide novel insights into the relevance of LPA receptors in FLS biology and as potential therapeutic targets for the treatment of RA.