A study of the uncharacterized serum inhibitors of hyaluronidase, first described half a century ago, was undertaken. Activity was measured against bovine testicular hyaluronidase using a microtiter-based assay and reverse hyaluronan substrate gel zymography. The predominant inhibitory activity was magnesium-dependent and could be eliminated by protease or chondroitinase digestion and by heat treatment. Kinetics of inhibition were similar against hyaluronidases from testis and snake and bee venoms. The inhibitor had no effect on Streptomyces hyaluronidase, indicating that inhibition was not through protection of the hyaluronan substrate. Inhibition levels in serum were increased in mice following carbon tetrachloride or interleukin-1 injection, inducers of the acute-phase response. Reverse zymography identified a predominant band of 120-kDa relative molecular size, with two bands of greater and one of smaller size. The predominant protein was tentatively identified as a member of the inter-alpha-inhibitor family. Inhibition was also observed using either purified inter-alpha-inhibitor or an inter-alpha-inhibitor-related 120-kDa complex. Inter-alpha-inhibitor, found in the hyaluronan-rich cumulus mass surrounding mammalian ova and the 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 2-5 min. Prompt increases in levels of serum hyaluronan occur in patients with shock, septicemia, or massive burns, increases that can be attributed, in part, to suppression of degradation by these acute-phase reactants, the inhibitors of hyaluronidase.