We developed a double-isotope autoradiographic method for the simultaneous measurement of the local cerebral metabolic rate for glucose (1CMRG) and index of regional acid-base status (rABI) in single brain slices using [2-14C]deoxy-D-glucose (DG) and 5,5-dimethyl-[2-14C]oxazolidine-2,4,dione (DMO). After iv isotope administration, paper chromatography separates plasma DMO from DG activity using a methanol-methylene chloride solvent system. Initial tissue autoradiograms depict regional DMO plus DG and DG metabolite distribution. After 14 days in a well-ventilated hood, 97.5 +/- 0.5% of all DMO is lost from tissue sections by sublimation, and a second autoradiogram depicts DG plus DG metabolite distribution. Retention of brain lipids does not alter beta-particle self-absorption, avoiding problems associated with isotope extraction with solvents. Autoradiograms are digitized and converted to isotope-content images. The second autoradiogram is used for 1CMRG computation. After subtracting the second regional isotope-content value from the first, the DMO content is obtained and used to compute rABI. Application of this method to normal animals yields expected values for 1CMRG and rABI. This method is amenable to whole-slice digitization and creation of functional images of 1CMRG and ABI followed by pixel-by-pixel correlations of the two variables, making this a potentially valuable tool for the investigation of the relationships between glucose metabolism and brain acid-base balance.