It has been shown that intravenously injected bovine testicular hyaluronidase reduces ischemic myocardial necrosis in animal models of myocardial infarction. The mechanism for the protective effect of hyaluronidase is unknown, but it has been suggested that this enzyme depolymerizes myocardial hyaluronic acid and thus improves interstitial transport. However, biochemical data showing that intravenous hyaluronidase depolymerizes myocardial substrates are lacking. The purpose of the present study was to test the ability of hyaluronidase to depolymerize myocardial hyaluronic acid. Thirty minutes after occlusion of the left anterior descending coronary artery, nine dogs were randomized to untreated (four dogs) or hyaluronidase-treated (five dogs; 5000 National Formulary Units/kg, iv, and 0.5 and 2.5 hours post occlusion) groups. The hearts were excised 4.5 hours postocclusion and the myocardium was homogenized. Myocardial bound hyaluronidase was heat inactivated, and hyaluronic acid was extracted following protease digestion. Myocardial hyaluronic acid content was determined colorimetrically and its molecular weight was studied by Sepharose 2B gel exclusion chromatography. There was no difference in hyaluronic acid recovery or molecular weight between the ischemic anterior and non-ischemic posterior halves of the hearts of both treated and untreated dogs. Hearts of the untreated dogs contained 3.44 ± 0.32 fig hyaluronic acid/mg protein, whereas hyaluronidase-treated hearts contained only 2.06 ± 0.23 /ig hyaluronic acid/mg protein (P< 0.01). The fraction of hyaluronic acid that chromatographed with molecular weight greater than 4 x 106daltons was 73 ± 2% in untreated and only 45 ± 5% in hyaluronidase-treated animals (P< 0.001). Therefore, treatment with hyaluronidase reduces both the amount and the molecular weight of myocardial hyaluronic acid in dogs with coronary artery occlusion. Circ Res 47: 88-95, 1980