N-type inactivation of voltage-gated K+ channels is essential for fine-tuning the electrical excitability of neurons. For some types of regulation, Cys residues in the N termini of α subunits play prominent roles. For example, C13 in KV1.4 channels makes the inactivation susceptible to redox state changes [1] and heme binding [2]. Since the endogenously produced gaseous messenger hydrogen sulfide (H2S) affects action potential width and frequency [3], we investigated if and how H2S affects N-type inactivation. Rat KV1.4 channels were expressed in HEK 293T cells, and currents were measured in the whole-cell patch-clamp configuration at 19-21°C. The H2S donor NaHS (1 mM) progressively removed inactivation within about 300 s. Even 200 µM NaHS was effective, and its potency increased with the NaHS solution aging. This suggested that polysulfides, which spontaneously form in NaHS solutions [4], were the active components. In fact, sodium polysulfides (Na2Sn) were about 1000-fold more active than NaHS, with the potency increasing with the number of sulfur atoms; even 100 nM Na2S4 efficiently slowed down inactivation. The reducing agent dithiothreitol (DTT, 1 mM) reversed this effect, suggesting Cys are targets. Consistently, KV1.4 mutant C13S was insensitive to 1 mM NaHS and 10 µM Na2S4. Mass spectrometry analysis of a peptide encompassing the first 61 residues of KV1.4, once exposed to NaHS and Na2S4, showed a mass increase corresponding to one sulfur atom whereas the C13S mutant peptide was unaffected. Rat KV3.4, another mammalian N-type inactivating K+ channel, exhibited faster inactivation (time constant at 50 mV, mean±SEM: 16.0±1.3 ms, n=8) than KV1.4 (84.4±2.8 ms, n=7). Exposure to 1 mM NaHS or 1 µM Na2S4 resulted in a rapid loss of inactivation and a substantial increase of peak current, both of which were reversible with application of 1 mM DTT. Unlike KV1.4, the N terminus of KV3.4 harbors two Cys (C6, C24). Removal of both Cys was required to eliminate the channel’s sensitivity to NaHS and Na2S4. In the presence of the reagents, inactivation of the single mutants C6S and C24S was slowed but not removed completely. The Cys-specific modifier DTNP (2 µM) and diamide-induced (10 µM) glutathionylation both removed inactivation in KV3.4 channels, and both Cys take part in the underlying mechanism. Marked slow-down of inactivation was observed in the whole-cell recording mode where the cytosol is exposed to the potentially oxidizing intracellular pipette solution. Cell-attached recordings after 10 min incubation of the cells with 10 µM Na2S4 or 100 µM diamide yielded similar results. In summary, H2S and polysulfides can impair N-type inactivation of KV1.4 and KV3.4 channels by targeting Cys residues in the N-terminal ball-and-chain domain and are therefore potential modifiers of neuronal electrical excitability.