The 2-deoxyglucose (2-DG) method of functional neuroanatomical mapping25 was re-examined in order to (1) obtain physical descriptions of the transfer constants K1 and k2, (2) estimate the changes of the 'lumped constant' with the condition of the experimental animals, and (3) examine the use of 3-O-methylglucose (3-O-MG) to estimate the fraction of unphosphorylated 2-DG in the tissue, and the value of the 'lumped constant'. The transfer constants K1 and k2 were shown to be simple exponential forms of the apparent permeability of the cerebral capillary endothelium to glucose and glucose analogs. The 'lumped constant' was shown to be influenced by any reduction of the ratio between glucose transport and glucose phosphorylation in the tissue, e.g. by hypoglycemia and increased glycolysis, while hyperglycemia and decreased glycolysis resulted in very minor changes of the 'lumped constant'. The glucose analog 3-O-MG was shown accurately to trace unphosphorylated 2-DG in brain and to be an index of the brain content of glucose and the regional value of the 'lumped constant'. In addition, 3-O-MG proved to be an accurate tracer of unphosphorylated 2-DG for experimental times as low as 10 min.