There is experimental and clinical evidence that i.v. injection of bovine testicular hyaluronidase (BTH) reduces the extent of necrosis during myocardial infarction. The fate of i.v. administered BTH has not been described. In this study, serum kinetics of BTH enzyme activity in dogs, rats and humans were determined. Tissue distribution of BTH was determined with an 125I-labeled preparation of purified BTH. Serum BTH activity initially decreased exponentially with half-life 2.0 +/- 0.1 min in dogs with coronary artery occlusion (n = 8; 500 U of BTH/kg); 3.2 min in humans with acute myocardial infarction (n = 2; 500 U of BTH/kg); and 3.2 +/- 0.3 min in rats (n = 5; 5,000 U of BTH/kg). In dogs BTH disappearance showed two distinct phases. After injection of high dose BTH (5,000 U of BTH/kg), during the first 7 min serum half-life of BTH was 2.1 +/- 0.2 min (n = 8), but increased to 9.4 min in later serum samples. After the injection of 125I-labeled BTH into the rat, protein-bound 125I disappeared from serum with a half-life (3.4 min) that is similar to the serum half-life of BTH enzyme activity (3.2 min). Twenty minutes after injection of 125I-labeled BTH, 30% of the label was recovered in the liver. It is concluded that BTH activity has a short serum half-life of less than 10 min in dogs, rats and humans. In the rat model, the disappearance of serum BTH activity results from physical removal of circulating BTH molecules rather than serum inhibition or inactivation of BTH enzymatic activity.