A method is described for the isolation and purification of glucose dehydrogenase from bovine liver in which Triton X-100 is used to release glucose dehydrogenase from the endoplasmic reticulum. This particular form of the enzyme contains a limited amount (1.5%) of cholesterol, fatty acids, triglycerides, and cholesterol esters. The Circular Dichroism data analysis indicates 52% alpha helix, 15% beta pleated sheet, and 33% random for this enzyme. The molecular weight is estimated at 235,000 and that of the subunits is 59,000 as determined by gel electrophoresis. The pH of maximum activity is 9.5 in 50 mm glycine buffer at 30 °C. At pH 7.5 and above, this enzyme catalyzes significant rates of glucose oxidation at physiologically meaningful concentrations of glucose. A steady-state analysis of glucose dehydrogenase kinetics is made at 30 °C (pH 8.0, 9.5, and 10) and 37 °C (pH 7.5 and 9.5). At pH 9.5 and 37 °C, Kia's for NAD and NADP are similar (5-6 ?m) and the Kb's for glucose and glucose 6-phosphate are 4.2 mm and 25.4 ?m, respectively. At pH 7.5 and 37 °C, the Kia's for NAD and NADP are 1.2 and 3-4 ?m, respectively, while the Kb values for glucose and glucose 6-phosphate are 4.8 mm and 3.0 ?m. There is no apparent product inhibition by NADH or NADPH. Similarly, the glucose-NAD reaction is not inhibited by either glucose 6-phosphate or by 1,5-gluconolactone. An overall model is presented for the role of glucose dehydrogenase based upon kinetic evidence. Glucose dehydrogenase is seen as a multifunctional protein involved in several catabolic sugar pathways in the endoplasmic reticulum. © 1982.