Brain glucose content is an important experimental variable that affects the value of the “lumped constant” of the 2-deoxyglucose method. The apparent volume of distribution in brain of the nonmetabolizable glucose analog, 3-O-methylglucose, depends only on the glucose content. From the kinetic constants of glucose transport and the apparent volume of distribution, we used autoradiography to calculate the regional glucose content of the normal rat brain. The regional glucose content varied only insignificantly in gray matter regions; the average glucose content of all rat brain slices examined was 4 μmol g⁻¹, with an average plasma glucose concentration of 8.6 mM. Regional values varied between 3.4 and 4.6 μmol g⁻¹. Thus, there is no reason to believe that the regional values of the lumped constant vary significantly in normal rat brains.