Akabayashi et al. made a valuable modification of the enzymatic methods from our laboratory for measuring 2-deoxyglucose and 2-deoxyglucose 6-phosphate. Their modified procedure eliminates glucose and glucose 6-phosphate by conversion to fructose-1,6-bisphosphate, thereby saving two analytical steps. However, the present report describes a limitation of this new elimination procedure which is due to its unexpected reversibility, and provides an easy way to circumvent this limitation, namely heating to destroy the reagent enzymes before proceeding. The final result is a more flexible analytical scheme that is capable of measuring 2-deoxyglucose and its phosphate down to extremely low levels in the presence of up to thousandfold higher glucose concentrations. The completeness of glucose elimination eliminates both the problem of contamination of available glucose-6-phosphate dehydrogenases with 6-phosphogluconate dehydrogenase, and also the effect of the presence in this same enzyme of a trace of glucose dehydrogenase activity, which is an apparent side reaction.