Sphingosine 1-phosphate (S1P) is a pleiotropic bioactive lipid that transmits potent signals through a family of G protein coupled receptors with resultant anti-apoptotic and pro-angiogenic effects. We have recently reported that lymphoblastoid B cell lines (LCLs) from rheumatoid arthritis (RA) patients are resistant to Fas-mediated cell death due to over-production of S1P, secondary to over-activity of sphingosine kinase-1 (SphK1). Here we investigated the signaling events that S1P triggers in those cells. Our results show that RA-derived LCLs display increased constitutive enzymatic activity of phosphatidylinositol 3-kinase (PI3K). Incubation of LCLs with a PI3K inhibitor wortmannin reversed PI3K over-activity and the resistance to Fas-mediated cell death. Incubation of RA LCLs with nanomolar concentration of S1P triggered exaggerated activation of both SphK and PI3K in RA LCLs compared to control cells. PI3K was mapped upstream of SphK, since wortmannin could block SphK activation by S1P. S1P signaling effect could be blocked by the G_i/G₀ protein inhibitor, pertussis toxin and by an inhibitor of S1P-receptor interaction, suramin. S1P receptor expression levels did not appear to be the cause of disparate S1P-triggered signaling, since LCLs from RA patients and their healthy twin controls did not show statistically significant differences in the expression levels of the five known S1P receptors, as determined by quantitative real time reverse transcription-polymerase chain reaction analyses. Thus, we conclude that Fas death signaling aberration in RA LCLs is caused by extracellular S1P, which triggers PI3K-dependent SphK over-activity through a G_i protein-coupled receptor-mediated signaling cascade.