Glycolipid transfer protein (GLTP) catalyzes the intermembrane transfer of lipids that have sugars beta-linked to either diacylglycerol or ceramide backbones, including simple glycosphingolipids (GSLs) and gangliosides. The present study provides a quantitative understanding of GLTP action involving bilayer vesicles that have high and low curvature stress, i.e., small and large unilamellar vesicles (SUVs and LUVs). When the GSL intervesicular transfer was monitored in real time using an established fluorescence resonance energy approach, the initial GSL transfer rates (v(0)) and net transfer equilibrium (K(eq)) were determined for GLTP-mediated transfer from SUVs and LUVs over the temperature range of 30-44 degrees C. v(0) exhibited a linear dependence with respect to varying GLTP concentrations (0-143 nM range) in SUVs and LUVs, suggesting a first order dependence on the GLTP bulk concentration. Thermodynamic parameters associated with the GLTP-GSL transition-state complex and GSL net transfer were determined from linear Arrhenius and van't Hoff plots, respectively. Although initial transfer rates were lower for LUVs than for SUVs, the activation energy barriers were higher for LUVs, while the Gibbs's free energy of the transition states were similar. The formation of a transition-state complex was predominantly enthalpy driven, whereas the net transfer of GSLs was mainly entropy driven. The rate-limiting step for GLTP action appeared to be the surface processes leading to the GLTP-GSL complex formation and release associated with a shuttle/carrier mode of action. Because surface processes leading to the GLTP-GSL complex formation were limiting for GLTP action with SUVs and LUVs, it was concluded that GLTP is likely to be a valuable tool to probe and manipulate GSL environments in membranes.