Hyaluronidase (hyaluronate 4-glycanohydrolase, EC 3.2.1.35) was purified from mouse testes by ion-exchange chromatography, Sephadex G-200 filtration and Con A-agarose affinity chromatography. The final preparation had 94-fold purity and 12.2 units spec. act. of the enzyme (unit of specific activity = mumol N-acetylglucosamine released/h per mg protein at 37 degrees C and pH 4.5). Hyaluronidase is relatively heat stable and loses 10-20% of its activity at 50-55 degrees C for 10 min. Ea for eat denaturation of enzyme is 42-45 kcal between 45 an 63 degrees C. The Michaelis constant of mouse testicular hyaluronidase is 1.1 mg/ml hyaluronic acid. Antibodies to the purified enzyme were produced in rabbits and showed a single precipitin line by Ouchterlony gel diffusion. Antiserum to hyaluronidase inhibited enzyme activity by 25%. Immunologically, mouse testicular hyaluronidase is species specific. Tissue extracts of mouse vital organs, except testes and epididymis did not react with the antisera, though nonspecific precipitation occurred between intestinal extracts and anti-hyaluronidase serum. Hyaluronidase was localized in testis sections by indirect immunofluorescence. A specific dark green fluorescence was localized on cell boundaries extending from spermatogonia to spermatids and appeared on the sperm acrosome. Cytoplasm of spermatogonia and spermatocytes showed light green fluorescence, whereas interstitial tissue was devoid of fluorescence.