A 'soluble carbonic anhydrase (carbonate hydro-lyase, Eq 4.2.1.1) has been purified to homogeneity from the membranods lateral wall (stria vascularis, spiral ligament, spiral prominence, and outer sulcus) of the guinea-pig inner ear. About 1% of the protein of the membranous lateral wall is carbonic anhydrase. The specific activity of the enzyme in hoinogenates of the lateral wall is 1.6-1.8 times that of whole blood; for homogenates of the components, stria vascularis and the fraction containing the spiral ligament, the specific activities are 0.9 and 2.0 times the specific activity of whole blood, MATERIALS AND METHODS All chemicals used in these studies were of reagent grade or better. 5-Dimethylaminonaphthalene-l-sulfonamide (DNSA) was from K & K Fine Chemicals, Plainview, N.Y., and the chromatographic resins were from Bio-Rad, Richmond, Calif.The animals were normal, colored guinea pigs of both sexes weighing 160-320 g. Animals were decapitated and their blood was collected in containers on ice. Blood from each animal was diluted 'oo or 6oo in deionized water at 4°. Temporal bones were dissected with surgical scissors and placed on ice within 1 min of death. Intact cochleas were then removed and immersed in a dissecting medium of 10 mM Tris-sulfate, 100 mM Na2SO4, pH 7.4 at 1-2°. Dissections were performed in the medium using a dissecting microscope. A sample of bony shell that covered the fourth turn was chipped away with a forceps and the remaining cochlear bone was removed, exposing the intact spiral of the membranous lateral wall, which was unwound (10). The basilar and tectorial membranes, the remaining cochlear epithelium, and a small amount of bone, comprising the "organ-of-Corti fraction," were scraped from the bony spiral with a finely pointed forceps. The auditory nerve was dissected from the modiolus to the internal auditory meatus. In separate experiments, the medulla oblongata was exposed and the left temporal bone was removed after the cranial nerves entering it were cut. Then the whole cochlear nucleus was dissected with a small spatula. All dissected tissues were rinsed in cold medium, placed in ground-glass homogenizers (0.2-ml nominal capacity; Kontes Glass Co., Vineland, N.J.), homogenized in 50 AM of deionized water for 15-30 min on ice, and diluted with water to the following volumes: 30 ,Ml/sample of cochlear bone containing 16 ,ug of protein, 2 ml/cochlear nucleus, 300 Ml/auditory nerve, 70Al/organ-ofCorti fraction from one cochlea, and 220 Al/membranous lateral wall from one cochlea. Each fraction, except cochlear nucleus and whole blood, contained tissue samples from more than one animal. Each experiment (Table 1) consisted of the analysis of one fraction. The average number of animals used per fraction can be determined from Table 1.Lyophilized tissue was used in experiments where the stria vascularis was separated from the spiral ligament fraction, because the use of fresh tissue caused loss of carbonic anhydrase into the medium when the fractions were separated. Fresh coc...