Clostridium perfringens is the Gram positive causative agent of human clostridial myonecrosis and food poisoning. We have identified an orphan response regulator, RevR, which has sequence identity to the PhoB family of response regulators and is predicted to contain a helix-turn-helix DNA binding motif. Importantly, a revR mutant is attenuated for virulence in the mouse myonecrosis model. The revR gene is situated downstream of genes encoding a putative phosphate uptake (pst) operon. Transcriptomic analysis showed that the pst genes were down-regulated in a revR mutant. Gel mobility shift experiments showed that RevR binds to the pst promoter regions, while DNase I footprinting demonstrated that the DNA sequence recognised by RevR had organisational and sequence similarity to Pho boxes from other bacteria. Bioinformatic analysis suggested that the CPE1757 orphan sensor histidine kinase, which we have designated as RevS, was a homolog of the PhoR sensor histidine kinase. Interaction between RevR and RevS was predicted using two different algorithms and subsequently was confirmed experimentally with two different methods. Phosphotransfer was demonstrated between RevS and RevR in vitro. Finally, we mutated the revS gene and showed that the resultant mutant was attenuated for virulence, to levels similar to that of the revR mutant. In conclusion, we have shown that RevR binds to Pho boxes, indicating that it is potentially part of a phosphate regulon, and we have identified its cognate sensor histidine kinase, RevS. We now have identified the two essential parts of a new regulatory network that is involved in the regulation of virulence in C. perfringens.