BACKGROUND. Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are lipid mediators that bind to G-protein-coupled receptors. In this study, signaling responses to 18:1 LPA and S1P were examined in parallel in three human prostate cancer cell lines: PC-3, Du145, and LNCaP. METHODS. Receptor expression was assessed by RT-PCR, Northern blotting, and immunoblotting. Cellular responses to mediators were studied by proliferation assays, phosphoprotein immunoblotting, and phospholipid metabolism assays. RESULTS. All cell lines express mRNA for both LPA and S1P receptors. PC-3 and Du145, but not LNCaP, proliferate in response to LPA and S1P. Epidermal growth factor (EGF), phorbol 12-myristate 13-acetate (PMA), LPA, and S1P induce activation of Erks in PC-3 and Du145; only EGF and PMA activate Erks in LNCaP. In Du145 and PC-3, Akt is activated by EGF, LPA, and S1P. Akt is constitutively active in LNCaP; EGF but not LPA or S1P stimulates further phosphorylation. FAK is phosphorylated in response to both LPA and S1P in PC-3 and Du145, but not in LNCaP. LPA and S1P stimulate phospholipase D (PLD) activity to varying extents in the different cell lines. Notably, both lipid mediators activate PLD in LNCaP. In Du145, LPA, but not S1P, activates PLD and enhances cellular production of LPA. CONCLUSIONS. Although both LPA and S1P induce signal transduction in all prostate cancer cell lines studied, a proliferation response is observed only when the Erk, Akt, and FAK pathways are activated. Other responses to the lipid mediators, such as PLD activation, likely contribute to other cellular outcomes. Prostate 69: 1493-1506, 2009. (C) 2009 Wiley-Liss, Inc.