Neural stem/progenitor cells are clonogenic in vitro and produce neurospheres in serum-free medium containing epidermal growth factor (EGF) and fibroblast growth factor (FGF2). Here, we demonstrate that lysophosphatidic acid (LPA) instigated the clonal generation of neurospheres from dissociated mouse postnatal forebrain in the absence of EGF and FGF2. LPA induced proliferation of cells which co-expressed Sca-1 antigen and AC133, markers of primitive hematopoietic and neural stem/progenitor cells. Clonal expansion of these cells induced by LPA was inhibited by diacylglycerol- pyrophosphate (DGPP), an antagonist of the LPA receptor subtypes LPA1 and LPA3. Moreover, Sca-1- and AC133-positive cells of these neurospheres expressed LPA1, LPA2, and LPA3, suggesting important roles for these LPA receptors in proliferation of neural progenitors. LPA induced neurospheres to differentiate on an adherent laminin/poly-L-ornithine matrix. In differentiating neurospheres, LPA receptors co-localized with betaIII-tubulin, nestin, and CNPase, but not with glial fibrillary acidic protein (GFAP), a marker of astrocyte lineage. Our results demonstrate for the first time that lysophosphatidic acid induces clonal neurosphere development via proliferation of AC133/Sca-1-positive stem cells by a receptor-dependent mechanism. This differentiation was characterized by the initial co-localization of neural specific antigens at sites of LPA receptor expression upon their interaction with the inducing agonist.