The most well-known role of Connexins (Cxs) is to communicate the cytoplasm of two adjacent cells forming a gap junction channel in the apposition zone. On the other hand, Cxs form the so called “hemichannels”, a fully functional voltage-gated non-selective ion channel, which present two gating mechanism called “slow” and “fast” gating. Hemichannels consist of 6 monomers, each one composed of four transmembrane domains. Despite the fact that hemichannels are voltage-activated, they lack the classical voltage-sensing domain described for canonical voltage-gated channels. Furthermore, the molecular determinants associated to the voltage activation in connexins hemichannels are still elusive. In this work we search for the molecular determinants associated to voltage detection in human Cx26 hemichannels. Using two electrode voltage-clamp in Xenopus laevis oocytes, we study how the steady-state conductance-voltage (G/V) relationship and kinetics of gating are affected by the neutralization of different charged residues located within the first transmembrane domain. We have found that Cx26 conductance-voltage curve shows an apparent number of gating charges (zδ) of 1.4 e0 for the slow gating mechanism. On the other hand, we obtained a zδ of 3.9 e0 for the fast gate, which is in agreement with the zδ of Cx26 gap junctions. A simple three-state closed-open1-open2 kinetic model is able to account for the channel voltage dependence. Furthermore, our data shows that neutralizing the K41 residue (K41N) increases the zδ; of the slow gate to 2.5 e0, and decreases the zδ of the fast gate to 2.3 e0.This work is supported by FONDECYT Grants 1110430 (R.L.), 1120802 (C.G.), 1130855 (A.M.); ANILLO Grant ACT1104 (C.G.); Beca para Estudios de Magister en Chile Ano 2014 (B.P.); CINV is a Millennium Institute supported by the Millennium Scientific Initiative of the Ministerio de Economia, Fomento y Turismo.