It has been reported recently that the phosphorylated form of the immunomodulator FTY720 activates sphingosine 1-phosphate G protein-coupled receptors [1,2]. Therefore, understanding the biology of this new class of receptors will be important in clarifying the immunological function of bioactive lysosphingolipid ligands. The S1P(4) receptor has generated interest due to its lymphoid tissue distribution. While the S1P(4) receptor binds the prototypical ligand, S1P, a survey of other lysosphingolipids demonstrated that 4D-hydroxysphinganine I-phosphate, more commonly known as phytosphingosine I-phosphate (PhS1P), binds to S1P(4) with higher affinity. Using radiolabeled SIP (S1(33)P), the affinity of PhS1P for the S1P(4) receptor is 1.6 nM, while that of SIP is nearly 50-fold lower (119 1 +/- 20 nM). Radiolabeled PhS1P proved to be superior to S1(33)P in routine binding assays due to improved signal-to-noise ratio. The present study demonstrates the utility of a novel radiolabeled probe, PhS1(33)P, for in vitro studies of the S1P(4) receptor pharmacology. (C) 2002 Elsevier Science (USA). All rights reserved.