BACKGROUND: Activation of voltage-gated potassium channels of the Kv7 (KCNQ) family reduces cellular excitability. These channels are therefore attractive targets for treatment of diseases characterized by hyperexcitability, such as epilepsy, migraine and neuropathic pain. Retigabine, which opens Kv7.2-5, is now in clinical trial phase III for the treatment of partial onset seizures. One of the main obstacles in developing Kv7 channel active drugs has been to identify compounds that can discriminate between the neuronal subtypes, a feature that could help diminish side effects and increase the potential of drugs for particular indications. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we have made a thorough investigation of the Bristol-Myers Squibb compound (S)-N-[1-(4-Cyclopropylmethyl-3,4-dihydro-2H-benzo, oxazin-6-yl)-ethyl]-3-(2-fluoro-phenyl)-acrylamide [(S)-2] on human Kv7.1-5 channels expressed in Xenopus laevis oocytes. We found that the compound was a weak inhibitor of Kv7.1. In contrast, (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. The enhancing effects of (S)-2 were critically dependent on a tryptophan residue in S5 also known to be crucial for the effects of retigabine, (S)-1 and BMS-204352. However, while (S)-2 did not at all affect a mutant Kv7.4 with a leucine in this position (Kv7.4-W242L), a Kv7.2 with the same mutation (Kv7.2-W236L) was inhibited by the compound, showing that (S)-2 displays a subtype-selective interaction with in the Kv7 family. CONCLUSIONS/SIGNIFICANCE: These results offer further insight into pharmacological activation of Kv7 channels, add to the understanding of small molecule interactions with the channels and may contribute to the design of subtype selective modulators.