The glucose analog, 2-deoxy-D-glucose (2DG), has been used widely for studying the initial steps in the metabolism of glucose by radio-isotope tracer methods and by 31P NMR. In the rat heart perfused with acetate/2DG (both 5 mM) plus insulin, trapping of phosphorus by 2-deoxy-D-glucose-6-phosphate (2DG6P) results in a steady state exhibiting high 2DG6P (55 mM) and low ATP concentrations but near-normal function, as observed in an earlier 31P NMR study. In order to understand how the 2DG6P concentration is stabilized, we studied the inhibition of a mammalian hexokinase by 2DG6P in vitro by a 31P NMR technique. Inhibition, previously unobserved, was found. It is similar to inhibition by G6P in that it is competitive with ATP and not competitive with 2DG, but the inhibition constant (1.4 mM) is much larger. The experimental protocol includes provisions for enzymatic destruction of stray inhibitors such as G6P. The results show that the high 2DG6P and low ATP concentrations found in the steady state of the perfused heart should strongly reduce the rate of phosphorylation of sugars by hexokinase.