Sphingosine-1-phosphate (S1P) is a naturally occurring polar phospholipid that exerts growth-factor--like effects in a number of cell types, potentially by endocrine, paracrine, and intracrine mechanisms. The biological actions of S1P are mediated in part by its specific binding to at least four members of the edg/lp family of G protein-coupled receptors (edg 1, 3, 6, and 8), and further regulated by the actions of specific cell membrane-bound lipid phosphate phosphatases (LPPs). Recent evidence has suggested that S1P may act as an osteoblast growth factor. In the current study, we demonstrate expression in osteoblastic cells of the S1P receptors edg 1, edg 5 and edg 8; the lipid phosphate phosphatases LPP-1, LPP-2, and LPP-3, and the S1P phosphatase mSPP1. S1P exerts dose-dependent mitogenic effects in cultures of primary rat osteoblastic cells and SaOS-2 cells. S1P also activates p42/44 mitogen-activated protein (MAP) kinases in osteoblastic cells, and the proliferative effects of S1P in these cultures are partially abrogated by PD-98059 and U-0126, specific inhibitors of p42/44 MAP kinase signaling. S1P-induced p42/44 MAP kinase activation in osteoblastic cells is dependent on functional Gi proteins and intracellular calcium fluxes, but not on protein kinase A, phospha-tidylinositol-3 kinase, or protein Kinase C. These data demonstrate (1) the expression by osteoblastic cells of several cell membrane-bound components of phospholipid signaling and (2) that S1P is a potent osteoblast mitogen, the proliferative action of which is mediated by a signaling pathway that involves Gi proteins, intracellular calcium, and p42/44 MAP kinases.