Objectives. We sought to describe the prevalence of a hyaluronidase gene and hyaluronidase production in 197 clinical isolates ofP. acnes; we assessed kinetics of hyaluronidase production in a subset of three isolates.Methods. The hyaluronidase gene was detected using polymerase chain reaction. Hyaluronidase production was detected by growing isolates on BHI agar containing 400 μg/mL hyaluronic acid and 1% albumin and flooding plates with 2 N glacial acetic acid to precipitate unbound hyaluronic acid, with a zone of clearing representing a positive phenotype. Hyaluronidase production kinetics were measured as a function of hyaluronic acid digestion over time in a liquid medium.Results. A hyaluronidase gene and hyaluronidase production were detected in 100 and 97% ofP. acnesisolates, respectively. Hyaluronidase production in liquid medium was detectable after 96 hours of growth.Conclusions. Hyaluronidase production is nearly universal amongP. acnesisolates. Three days appear to be required for significant hyaluronidase production in a liquid medium. Detection of hyaluronidase in tissue specimens may be a strategy to differentiateP. acnesinfection from colonization whenP. acnesis isolated from a clinical specimen.