In vivo binding of human inter-alpha-trypsin inhibitor to hyaluronate was investigated by immunoelectrophoretic techniques. Pathological synovial fluids and follicular fluids both contain high concentrations of soluble hyaluronate. Heavy chain epitopes of inter-alpha-trypsin inhibitor were firmly associated with the hyaluronate in synovial fluid and follicular fluid. The hyaluronate-bound inter-alpha-trypsin inhibitor epitopes did not cross-react immunologically with bikunin. Several hyaluronate-bound inter-alpha-trypsin inhibitor fragments with molecular masses in the range 120,000-30,000 Da were demonstrated by immunoblotting. Heavy chain 1 of inter-alpha-trypsin inhibitor was shown to associate with hyaluronate by amino acid sequence analysis of isolated hyaluronate-bound proteins. These data indicate that in vivo metabolism of inter-alpha-trypsin inhibitor takes place in pathological synovial fluid and in ovarian follicular fluid shortly before ovulation.