The pathogenesis of Streptococcus pyogenes infection, especially toxic shock-like syndrome (TSLS), is still not fully understood; however, the exoproteins have been considered to play a role. We analyzed the culture supernatant proteins (exoproteins) from a TSLS-related isolate belonging to M3 serotype S. pyogenes by two-dimensional gel electrophoresis and characterized a single protein spot by using BLAST database. We cloned the gene of this protein and named it sdalpha, which was similar to the deoxyribonuclease (DNase) sdc of S. equisimilis. We showed that the recombinant protein from the sda gene had DNase activity. By polymerase chain reaction, we found that the sda gene was present in most clinically isolated S. pyogenes including TSLS-related isolates. We thus conclude that Sdalpha is a new DNase of S. pyogenes.