N,N-dimethyl-D-erythro-sphingosine (DMS) is an N-methyl derivative of sphingosine and an inhibitor of protein kinase C (PKC) and sphingosine kinase (SK). In the present study, we examined the effects of DMS on intracellular Ca2+ concentration, pH, and glutamate uptake in human 1321N1 astrocytes. DMS increased intracellular Ca2+ concentration and cytosolic pH in a concentration-dependent manner. Pretreatment of the cells with the Gi/o protein inhibitor PTX and the PLC inhibitor U73122 had no obvious effect. However, removal of extracellular Ca2+ with the Ca2+ chelator EGTA or depletion of intracellular Ca2+ stores with thapsigargin impeded the DMS-induced increase of intracellular Ca2+ concentration. Pretreatment of cells with NH4Cl or monensin reduced the DMS-induced Ca2+ increase. However, inhibition of the DMS-induced Ca2+ increase with BAPTA did not influence the DMS-induced pH increase. DMS also inhibited glutamate uptake by the 1321N1 astrocytes in a concentration-dependent manner. It also increased intracellular Ca2+ and pH in PC12 neuronal cells. Our observations on the effects of DMS on 1321N1 astrocytes and PC12 neuronal cells point to a physiological role of DMS in the brain.