The Staphylococcus aureus 8325-4 hyaluronate lyase gene (hysA) was identified after detecting hyaluronate lyase activity expressed by phages from a genomic library. The hysA open reading frame, capable of encoding a protein of 91 980 Da, was identified by Tn5 mutagenesis and nucleotide sequencing. HysA shares 35 and 36% amino acid sequence identity with group B streptococcal hyaluronate lyase and pneumococcal hyaluronidase, respectively. A 94-kDa protein was expressed in Escherichia coli minicells, a result consistent with the coding capacity of hysA. Identification of the S. aureus 8325-4 hyaluronate lyase gene will allow the regulation of this putative virulence determinant to be studied.