The inhibitors of hyaluronidase present in mammalian sera, first described half a century ago, have remained uncharacterized. Because of increased interest in hyaluronidases and their hyaluronan substrate, a study of these inhibitors was undertaken recently. The predominant serum inhibitor is magnesium-dependent and is eliminated by protease or chondroitinase digestion, and by heat. Kinetics of inhibition are similar against hyaluronidases from testis, snake and bee venom. The inhibitor has no effect on Streptomyces hyaluronidase; indicating inhibition is not through protection of the hyaluronan substrate. Circulating inhibition levels are increased in mice following carbon tetrachloride or interleukin-1 injection, inducers of the acute-phase response. Reverse hyaluronan gel zymography reveals a predominant band of 120 kDa relative molecular size. Additional studies indicate that the inhibitor resembles a member of the Kunitz type inter-alpha-inhibitor family. Inhibition of hyaluronidase activity is observed using purified inter-alpha-inhibitor and is reversed by antibodies specific for inter-alpha-inhibitor. This molecule, found in the hyaluronan-rich cumulus mass surrounding mammalian ova and the pericellular coat of fibroblasts and mesothelial cells, may function to stabilize such matrices by protecting against hyaluronidase degradation. Turnover of circulating hyaluronan is extraordinarily rapid, with a half-life of two to five min. Prompt increases in levels of serum hyaluronan occur in patients with shock, septicemia or massive burns, increases that may be partly attributed to suppression by these acute phase reactants of the constant and rapid rates of hyaluronan degradation by hyaluronidase. A literature survey of other hyaluronidase inhibitors is also presented.