Cystathionine b-synthase (CBS) catalyzes the condensation of serine and homocysteine to cystathionine, which represents the committing step in the transsulfuration pathway that converts methionine into cysteine. CBS is unique in being a pyridoxal phosphate-dependent enzyme that has a heme cofactor. The activity of CBS under in vitro conditions is responsive to the redox state of the heme, which is distant from the active site and has been postulated to be a redox sensor. The heme in CBS is unusual, it is six-coordinate, low spin, and contains cysteine and histidine as axial ligands. In this study, we have assessed the redox behavior of human CBS by cyclic voltammetry. Human dimeric CBS, a variant lacking the C-terminal regulatory domain, showed a quasi-reversible response on a bare platinum electrode, with anodic and cathodic peaks at ^_0.316 ± 0.017 V and ^_0.457 ± 0.006 V versus the normal hydrogen electrode (NHE), respectively. The normal reduction potential was estimated to be ^_0.341 ± 0.006 V vs NHE at pH 6.46. These results are compatible with the available crystal structural data that show that the heme in CBS is surface exposed and lies within a relatively electropositive environment. In addition to heme, CBS has an oxidoreductase motif (CXXC) that can exist in the reduced dithiol or oxidized disulfide state. Similar electrochemical profiles were obtained with CBS that had been treated with N-ethylmaleimide to block the thiols. The value of the redox potential implies that the heme in CBS could be sensitive to changes in the ambient reduction potential. Thus, in order to test if the heme could be reduced enzymatically, we incubated the enzyme with aliquots of rat liver extract in the presence of flavins (FAD/FMN) and pyridine nucleotides under anaerobic conditions. However, although the heme could indeed be reduced with dithionite, we did not observe NAD(P)H-dependent reduction of the heme, suggesting that the heme in CBS cannot be reduced by flavoproteins.