A triple-tracer autoradiographic technique is described that permits the simultaneous measurement of cerebral blood flow, glucose consumption, and protein synthesis using 131I-iodoantipyrine (131I-IAP), [14C]deoxyglucose ([14C]DG), and 3H-amino acids as radioactive tracers. Autoradiographic differentiation between isotopes was performed by taking advantage of different half-lives, solubility of labeled tracers in a wash solution, and sensitivity of the photographic material to disintegrations of the radionuclides. Blood flow autoradiograms using 131I-IAP were obtained by immediate exposure of brain sections to Kodak NMB film for 24 h. During 131I autoradiography contamination by 3H was absent and by 14C was negligible at tissue concentrations of less than 0.45 microCi/g brain tissue. After complete decay of 131I, reexposure of brain sections to Kodak NMB film for 2 weeks provided autoradiograms that stemmed exclusively from 14C disintegrations without contamination by either 131I or 3H and that represented regional glucose utilization. Brain sections were then wash-incubated for 12 h to remove [14C]DG, [14C]DG-6-phosphate, and free 3H-amino acids from the tissue, and exposed to 3H-sensitive LKB Ultrofilm for 2 weeks for autoradiography of 3H-amino acid incorporation into proteins. 14C radioactivity remaining in the tissue section after wash-incubation was determined by exposing sections again for 2 weeks to Kodak NMB film; the resulting contribution to the blackening of 3H-autoradiograms was corrected for by means of digital subtraction using an image-processing system. The triple-tracer autoradiographic technique was validated in rats under various physiological and pathophysiological conditions. In intact animals extinction correction was necessary only for 3H-autoradiograms. Under pathophysiological conditions, however, significant contamination of 131I by 14C occurred in regions with low blood flow and increased glucose utilization rate; this also required correction by digital subtraction. The interpretation of triple-tracer autoradiographic results is limited by the same restrictions as single-tracer autoradiography, but the simultaneous assessment of the three parameters considerably facilitates the interpretation of the flow/metabolic relationship, particularly under pathological conditions.