To examine intracellular signaling of human S1P3 (hS1P3), a sphingosine 1-phosphate (S1P) receptor in plasma membrane, hS1P3 DNA was transfected into RH7777 rat hepatoma cell line, and the inhibition of forskolin-induced cAMP accumulation and activation of MAP kinases by S1P were tested. In hS1P3 transformants, S1P inhibited forskolin-induced activation of adenylyl cyclase activity by about 80% and activated MAP kinases in dose-dependent and pertussis-toxin (PTX) sensitive manners. In oocytes expressing hS1P3 receptor, S1P evoked Cl⁣ conductance. These data suggested that PTX-sensitive G proteins are involved in hS1P3-mediated signaling, especially the positive action of S1P in cell proliferation. The potential advantages of rat hepatoma cells for the research of sphingosine 1-phosphate receptor are discussed.