The hyaluronate lyase (hyaluronidase) gene from Propionibacterium acnes was cloned and sequenced. The gene was isolated on an EcoRI-generated 3-kb piece of DNA. Expression was less in Escherichia coli than in P. acnes; in E. coli, active enzyme was only cell associated and not secreted. The gene is 2256-pb long and codes for a protein of 82 kDa. Amino terminal sequencing of the protein of the partially purified gene indicated the presence of a 32-amino-acid leader sequence. The leader sequence showed a membrane-spanning domain and all of the features usually associated with the leader for a secreted protein. The amino acid sequence is predicted to share homology with the hyaluronidase enzymes from Streptococcus pneumoniae, Streptococcus agalactiae, and Staphylococcus aureus. A potential hyaluronate-binding domain was identified and antibody against this domain was inhibitory to the enzyme.