Clostridium perfringens causes clostridial myonecrosis or gas gangrene in humans. C. perfringens produces many toxins and enzymes that aid in destroying human tissues and acquiring nutrients. To coordinate virulence-related gene expression, the VirSR two-component regulatory system regulates the vrr gene, which encodes a small regulatory RNA (sRNA) called VR-RNA. This sRNA controls the expression of many genes, including α-toxin gene, plcand κ-toxin gene, colA. However, detail of the mechanism of regulation is not clear, and it has been thought that more complicated regulatory networks are also involved.To identify a new putative regulator, a chromosome library was constructed and used to transform a gas gangrene isolate, strain 13. One transformant showed stronger α-toxin activity. Northern blot analysis showed that this strain had increased transcription of plc and colA. Sequence analysis showed that the insert encoded the CPE0205 gene and a sRNA, which was designated as VirY. Both CPE0205 and VirY were found to activate plc and colA, with a CPE0205 and VirY-double mutant showing less α-toxin activity than the wild type, but not as reduced as a VR-RNA mutant. Interestingly, expression of CPE0205 and VirY were found to be affected by VirSR. Network analysis using Microarray data performed under different culture conditions verified that VirY was involved in a complex network associated with toxin gene regulation. Although CPE0205 and VirY were demonstrated to regulate plc and colA transcription, the precise mechanism of regulation is as yet unknown. Work is currently underway to elucidate this novel regulatory mechanism.