The human ether-a-go-go related gene (HERG), encodes the pore forming a-subunit of the rapid delayed rectifier K⁺ channel which is central to the repolarisation phase of the cardiac action potential. HERG K⁺ channels have unusual kinetics characterised by slow activation and deactivation, yet rapid inactivation. The fourth transmembrane domain (S4) of HERG, like other voltage-gated K⁺ channels, contains multiple positive charges and is the voltage sensor for activation. In this study, we mutated each of the positively charged residues in this region to glutamine (Q), expressed the mutant and wild type (WT) channels in Xenopus laevis oöcytes and studied them using two-electrode voltage clamp methods. K525Q channels activated at more hyperpolarised potentials than WT, whereas all the other mutant channels activated at more depolarised potentials. All mutants except for R531Q also had a reduction in apparent gating charge associated with activation. Mutation of K525 to cysteine (C) resulted in a less dramatic phenotype than K525Q. The addition of the positively charged MTSET to K525C altered the phenotype to one more similar to K525Q rather than WT. Therefore it is not charge per se, but the specific lysine side chain at position 525, that is crucial for stabilising the closed state. When rates of activation and deactivation for WT and mutant channels were compared at equivalent total (chemical + electrostatic) driving forces, K525Q and R528Q accelerated activation but had no effect on deactivation, R531Q slowed activation and deactivation, R534Q accelerated activation but slowed deactivation and R537Q accelerated deactivation but had no effect on activation. The main conclusions we can draw from this data are that in WT channels K525 stabilises the closed state, R531 stabilises the open state and R534 participates in interactions that stabilise pre-open closed states.