The validity of optical density ratios used in [14C]2-deoxyglucose neuroanatomical mapping experiments is evaluated by comparing local cerebral glucose utilization (LCGU) and optical density (OD) ratios in the same animals. OD ratios are calculated by dividing the optical density of different gray matter structures by the optical density of a single white matter structure in each animal. OD ratios are linearly related to LCGU within a given animal including stimulated, highly activated regions. Anesthesia profoundly affects the relationship between LCGU and OD ratios, however, showing that OD ratios do not provide an accurate index of LCGU between animals in different physiological states. Anesthesia had only a slight effect on OD ratios, however, indicating that OD ratios may be helpful in assessing whether structures are functionally activated between animals in different physiological states.