The K(+) channels encoded by the human Ether-a-gogo Related Gene-1 (hERG1) are crucially involved in controlling heart and brain excitability and are selectively influenced by reactive oxygen species (ROS). To localize the molecular regions involved in ROS-induced modulation of hERG1, segmental exchanges between the ROS-sensitive hERG1 and the ROS-insensitive bovine ether-a-gogo gene (bEAG) K(+) channels were generated, and the sensitivity of these chimeric channels to ROS was studied with the two-microelectrode voltage-clamp technique upon their expression in Xenopus oocytes. Substitution of the S(5)-S(6) linker of hERG1 with the corresponding bEAG region removed channel sensitivity to ROS, whereas the reverse chimeric exchange introduced ROS sensitivity into bEAG. Mutation of each of the two hERG1 histidines at positions 578 and 587 within the S(5)-S(6) linker generated K(+) channels insensitive to modulation by ROS. In addition, the two iron chelators desferrioxamine (1 mm) and o-phenanthroline (0.2 mm) significantly inhibited hERG1 outward K(+) currents and prevented hERG1 inhibition induced by the ROS-scavenging enzyme catalase (1000 units/ml). Finally, the hERG1-inhibitory effect exerted by the iron chelators was prevented by the hERG1 H578D/H587Y double mutation. Collectively, the results obtained suggest that histidines at positions 578 and 587 in the S(5)-S(6) linker region of hERG1 K(+) channels are crucial players in ROS-induced modulation of hERG1 K(+) channels.