Three kinds of plasma membranes in rat islet homogenate were fractionated on sucrose gradient centrifuged at 70000g (F₁ : d²⁰=1.14-1.18, F₂ : d²⁰=1.06-1.12, F₃ : d²⁰=1.02-1.05). Every fraction indicated both enzyme activities of 5'-nucleotidase and ATPase which were typical marker enzymes in pancreatic plasma membrane, and the D-[5-³H] glucose binding with them were dose-dependently inhibited by D-glucose, but not by L-glucose, D-galactose and 3-O-methyl-D-glucose. The D-[5-³H] glucose binding with only F₂ fraction was inhibited by D-mannose and D-fructose which could stimulate insulin release (60%), phlorizin inhibited remarkably the binding with F₃ fraction (68%), however, the D-[5-³H] glucose binding with either F₁ or F₂ fraction was slightly inhibited by preincubation with alloxan (0.2 mg/ml, 37°, 10min) (F₁ : 29%, F₂ : 39%) and that with F₃ fraction was not effective (4%). It was found that the insulin release from the B granules which distributed in the pellet fraction on sucrose gradient centrifuged at 70000g, was induced by the interaction with F₂ fraction. Each maximal capacity of glucose binding site in F₁, F₂ and F₃ fractions was estimated at 3.2, 1.0 and 3.2 μmol/mg membrane protein, respectively, from those Scatchard's plots for the glucose binding. Consequently, it was estimated that the membrane which associated with insulin release was contained in F₂ fraction among three fractions from the islet homogenate.