We recently demonstrated that elevation of intracellular glucosylceramide (GlcCer) levels results in increased functional Ca2+ stores in cultured neurons, and suggested that this may be due to modulation of ryanodine receptors (RyaRs) by GlcCer (Korkotian, E., Schwarz, A., Pelled, D., Schwarzmann, G., Segal, M. and Futerman, A. H. (1999) J. Biol. Chem. 274, 21673-21678). We now systematically examine the effects of exogenously added GlcCer, other glycosphingolipids (GSLs) and their lyso-derivatives on Ca2+ release from rat brain microsomes. GlcCer had no direct effect on Ca2+ release, but rather augmented agonist-stimulated Ca2+ release via RyaRs, through a mechanism that may involve the redox sensor of the RyaR, but had no effect on Ca2+ release via inositol 1,4,5-trisphosphate receptors. Other GSLs and sphingolipids, including galactosylceramide, lactosylceramide, ceramide, sphingomyelin, sphingosine 1-phosphate, sphinganine 1-phosphate, and sphingosylphosphorylcholine had no effect on Ca2+ mobilization from rat brain microsomes, but both galactosylsphingosine (psychosine) and glucosylsphingosine stimulated Ca2+ release, although only galactosylsphingosine mediated Ca2+ release via the RyaR. Finally, we demonstrated that GlcCer levels were approximately 10-fold higher in microsomes prepared from the temporal lobe of a type 2 Gaucher disease patient compared with a control, and Ca2+ release via the RyaR was significantly elevated, which may be of relevance for explaining the pathophysiology of neuronopathic forms of Gaucher disease.