Sphingosine-1-phosphate (S1P) is a bioactive lipid that affects a variety of cellular processes through both its actions as a second messenger and via activation of a family of G protein-coupled receptors (S1P(1-5)). The study of S1P receptor pharmacology, particularly S1P(4), has been hindered by the lack of high-affinity radioligands with good specific activity. The studies presented herein characterize [(3)H]DH-S1P as a stable, high-affinity radioligand for S1P(4) pharmacology. Using a transfected Ba/F3 cell line selected for high hS1P(4) surface expression, we compared the consequences of different cellular backgrounds and commercial sources of sphingophospholipids on S1P(4) characterization. The development and subsequent use of the assay described has enabled us to extensively and definitively characterize the pharmacology of the human S1P(4) receptor.