The continuous delivery of glucose to the brain is critically important to the maintenance of normal metabolic function. However, elucidation of the hormonal regulation of in vivo cerebral glucose metabolism in humans has been limited by the lack of direct, noninvasive methods with which to measure brain glucose. In this study, we sought to directly examine the effect of insulin on glucose concentrations and rates of glucose transport/metabolism in human brain using 1H-magnetic resonance spectroscopy at 4 Tesla. Seven subjects participated in paired hyperglycemic (16.3 ± 0.3 mmol/1) clamp studies performed with and without insulin. Brain glucose remained constant throughout (5.3 ± 0.3 μmol/g wet wt when serum insulin = 16 ± 7 pmol/1 vs. 5.5 ± 0.3 μmol/g wet wt when serum insulin = 668 ± 81 pmol/1, P = NS). Glucose concentrations in gray matter-rich occipital cortex and white matter-rich periventricular tissue were then simultaneously measured in clamps, where plasma glucose ranged from 4.4 to 24.5 mmol/1 and insulin was infused at 0.5 mU · kg-1 · min-1. The relationship between plasma and brain glucose was linear in both regions. Reversible Michaelis-Menten kinetics fit these data best, and no differences were found in the kinetic constants calculated for each region. These data support the hypothesis that the majority of cerebral glucose uptake/metabolism is an insulin-independent process in humans.