Carbonic anhydrase was localized at the light microscope level using labeled inhibitor autoradiography. Laying hens, 10-day-old chicks and adult female Japanese quail received injections of 1-25 µC 3H-acetazolamide/g body weight. After 3 hr (quail) and 24 hr (hen and chick), 2-mm3 pieces of tissue were removed under anesthesia, frozen at –160°C, freeze-dried, fixed with OsO4 vapor, embedded in Epon, sectioned at 1 µ and covered with NTB-2 emulsion. The following results validate the method: (a) the concentration of acetazolamide was 100 times greater in red blood cells than in plasma 24 hr after injection in the hen, indicating that free and loosely bound acetazolamide was rapidly cleared from the body; (b) no significant amounts of label were found in muscle, cartilage, adipose tissue and nuclei, which have no carbonic anhydrase activity; (c) except for the pancreas, there was good agreement among the grain counts for the different classes of birds; and (d) the autoradiographic localizations were in general agreement with concepts of tissue function. Carbonic anhydrase was localized in: the proximal, distal, collecting tubules and the mesangium of the kidney; the acinar, but not the centroacinar, cells of the pancreas; the tubular glands and the columnar cells of the shell gland; the lining cells, but not the zymogenic cells, of the proventriculus; erythrocytes; erythroblasts; heterophils; and lymphoid nodules, but not in fat and interstitial tissue of bone marrow.