Human ether à go-go (hEAG1) voltage-gated potassium channels play an important role in neuronal signaling. hEAG1 proteins harbor a comparably large number of cysteine residues (19 per subunit as compared to 10 for Kv1.5), which could be readily modified by reactive oxygen species and free radicals liberated during neuronal activity. Here we elucidated the molecular mechanisms by which hEAG1 channel function is affected by sulfhydryl modification. hEAG1 was expressed in Xenopus oocytes and currents were recorded in the inside-out patch-clamp configuration. Application of the sulfhydryl-oxidizing reagent 5,5`- dithiobis(2-nitro-benzoic acid) (DTNB, 20 mM) to hEAG1 channels resulted in a biphasic decrease of ion current. Assayed at +40 mV, current decreased progressively with time constants of 132 s and 37091 s (n=5) with a contribution of the rapid component of 626%. In particular, the fast component was associated with a right-shift in the current-voltage relationship of about 30 mV, leading to a stronger contribution of the rapid component close to activation threshold (804% at -40 mV). To identify those residues sensitive to DTNB-mediated modification, we examined effects of DTNB on mutant channels in which one or more cysteines were substituted by alanines. A hEAG1 channel in which all cysteines of the N- and C-terminal domains were eliminated was insensitive to DTNB. The rapid effect of DTNB was abolished by concurrent replacement of three cysteines (C128, C145 and C214) in the cytosolic N-terminal domain. Re-introduction of single cysteine residues in a Cys-less background revealed that C145 and C214 are responsible for a rapid current reduction upon DTNB exposure to 406 and 618% (n=4–5), respectively. Cysteines at position C532 and C562 are responsible for slow complete loss of channel function developing with time constants of 50792 and 15030 s (n=5), respectively. We thus identified four functionally important cysteine residues in the cytosolic N- and C-terminal domains of hEAG1 channels. Notably, C532 is located in the linker between S6 and the cyclic nucleotide-binding domain (C-linker). At the homologous position in the cardiac hERG1 channel there is a conserved cysteine (C723), which is also responsible for the channel's oxidation sensitivity.