The effects of lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) on T cell expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), the diphtheria toxin (DT) receptor, were investigated in the Tsup-1 cultured line of human CD4+ 8+ 3low T lymphoblastoma cells. Tsup-1 cells bear endothelial differentiation gene (edg)-2 and -4-encoded G protein-coupled receptors (GPCRs) for LPA and Edg-3 and -5 GPCRs for S1P. Suppression by DT of Tsup-1 cell protein synthesis was enhanced by LPA and S1P, with lipid structural specificity similar to that required for their recognition by Edg receptors. LPA and S1P increased the Tsup-1 cell level of immunoreactive HB-EGF, and neutralizing antibodies to HB-EGF inhibited LPA and S1P enhancement of Tsup-1 cell susceptibility to DT. Stabilized transfection of Tsup-1 cells with a combination of plasmids encoding Edg-2 plus -4 antisense mRNA suppressed the levels of Edg-2 and -4, but not Edg-3 and -5, in Western blots and reduced in parallel the increments in HB-EGF and susceptibility to DT evoked by LPA but not S1P. Similar transfection with Edg-3 plus -5 antisense plasmids suppressed Tsup-1 cell levels of immunoreactive Edg-3 and -5, but not Edg-2 or -4, and concurrently reduced S1P-, but not LPA-, induced Tsup-1 cell increases in both HB-EGF and susceptibility to DT. Edg GPCR-mediated LPA and S1P enhancement of T cell sensitivity to DT, thus, may be attributable to increased expression of the DT receptor HB-EGF.