Objective: Voltage-gated potassium channels of the Kv7 (KCNQ) family are the molecular basis of the M-current that plays a key role in the regulation of neuronal excitability. Kv7.2/3 heteromers localize to the axon initial segment (AIS) of pyramidal neurons of adult rat hippocampus and in cultured hippocampal neurons. This localization is dependent on an ankyrin-G-binding motif in the channels' C-termini. Noteworthy Kv7.3 appears to be the major determinant of M channel localization to the AIS. Activation M-channels reduces cellular excitability. These channels are therefore attractive targets for treatment of diseases characterized by hyperexcitability, such as epilepsy, migraine and neuropathic pain. One of the main challenges in developing drugs that target Kv7 channels is to identify compounds that can discriminate between the subtypes, a prerequisite for minimizing side effects and improve the use of drugs for particular indications. Methods: We investigated the effect of several compounds such as (S)-N-[1-(4-Cyclopropylmethyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-ethyl]-3-(2-fluoro-phenyl)-acrylamide [(S)-2] or N-(6-Chloropyridin-3-yl)-3,4-difluorobenzamide (ICA-27243) on human Kv7.1-5 channels expressed in Xenopus laevis oocytes. Results: (S)-2 efficiently opened Kv7.2-5 by producing hyperpolarizing shifts in the voltage-dependence of activation and enhancing the maximal current amplitude. Further, it reduced inactivation, accelerated activation kinetics and slowed deactivation kinetics. The mechanisms of action varied between the subtypes. We found that for both Kv7.2 and Kv7.4, ICA-27243 does not depend on a tryptophan residue in the fifth transmembrane segment known to be critical for the effect of retigabine, indicating that the compound binds to a different site in the channels. Conclusion: These results offer insight into pharmacological activation of Kv7 channels which adds to the understanding of small molecule interactions with the channels and may contribute to the design of subtype selective modulators.