AbstractBasic helix‐loop‐helix (bHLH) transcription factors including Twist1 and E2a proteins regulate essential processes. These factors bind DNA as homo‐ or heterodimers and the choice of binding partners determines their functional output. To investigate potential regulators of bHLH dimerization, cells were exposed to the oxidative agent hydrogen peroxide (H2O2). Western blot analysis in the presence or absence of reducing agents, revealed that H2O2 induces the rapid formation of an intermolecular disulfide bond between Twist1 homodimers and Twist/E2a proteins heterodimers. The disulfide bond is first observed between Twist1 homodimers at 25 mM H2O2 and between Twist1 heterodimers at 75 mM H2O2. This response is dependent upon cell density as H2O2 did not induce disulfide bridge formation between bHLH proteins in cells seeded at high density. In the presence of E proteins, the formation of Twist1/E2a proteins heterodimers is favored over Twist1 homodimers, identifying an oxidative stimulus as an important factor in modulating binding partner specificity. We further demonstrated that a cysteine residue located at the C‐terminus of Twist1 and E2a proteins is involved in this response. Disulfide bond formation between Twist1 homodimers significantly reduced its ability to interact with two of its binding partners, Runx2 and HDAC4, indicating that disulfide dimerization in response to H2O2 has functional significance. These data support the conclusion that disulfide bond formation in response to an oxidative stimulus contributes to Twist1 homo‐ and heterodimerization and raises the possibility that the redox status of a cell may represent an important step in bHLH transcriptional regulation. J. Cell. Biochem. 109: 417–424, 2010. © 2009 Wiley‐Liss, Inc.