PefR is a heme-binding transcriptional regulator found in Streptococcus pyogenes, a β-hemolytic pathogen that can be fatal. Heme binding results in the release of the protein from DNA and allows for the transcription of a proposed heme efflux system. The overall goals of this work were to devise an effective protocol to purify DNA-free PefR, verify the stoichiometry of heme binding, and identify the axial ligand. Strep-tagged PefR was purified by a variety of protocols, the most effective of which utilized high salt concentrations in the buffers and an ion-exchange column. Based on UV/visible studies of PefR, we suggest that heme binds via Cys109. Homology models place Cys109 in a region that several MarR proteins utilize to bind their ligands, supporting our hypothesis.