As observed for depolarisation-activated cation channels, the fourth transmembrane segment (S4) in hyperpolarisationactivated HCN channels displays a distinct stripe of positively charged residues. These positively charged residues are believed to form a charged peptide particle that senses changes in membrane voltage, leading to channel gating. The voltage-sensing mechanisms of S4 in depolarisation-activated cation channels have been extensively studied (Bezanilla, 2000; Cohen et al., 2003). However, mechanistic studies on the S4 voltage-sensor of hyperpolarisationactivated cation channels have only recently started to be defined.To address this lack of definition, the substituted-cysteine accessibility method was used to topologically map residue positions along the HCN1 S4 according to their accessibility to the membrane impermeant, cysteine(Cys)-specific reactive reagent [2-(trimethylammonium) ethyl] methane thiosulphonate (MTSET). An MTSET-resistant HCN channel background (HCN-R) was created by removing endogenous MTSET-reactive Cys residues. Consequently, only specified, substituted-Cys in the HCN-R background would be reported by MTSET reactivity. The resulting pattern of MTSET reactivity for the substituted-Cys revealed distinct environmental changes around the S4 of the HCN channel during membrane hyperpolarisation and depolarisation (i.e. during channel gating and closing). Based on these data, existing models of voltage-sensing movements of the S4 (primarily derived from studies on depolarisation-activated cation channels) do not comply with the mammalian hyperpolarisation-activated HCN channels. New models were explored to reconcile the observed MTSET accessibility of HCN Cys-substituted residues in this study, providing pertinent voltage-sensing mechanisms in these channels.