Voltage-gated potassium (Kv) channels enable potassium efflux and membrane repolarization in excitable tissues. Many Kv channels undergo a progressive loss of ion conductance in the presence of a prolonged voltage stimulus, termed slow inactivation, but the atomic determinants that regulate the kinetics of this process remain obscure. Using a combination of synthetic amino acid analogs and concatenated channel subunits we establish two H-bonds near the extracellular surface of the channel that endow Kv channels with a mechanism to time the entry into slow inactivation: an intra-subunit H-bond between Asp447 and Trp434 and an inter-subunit H-bond connecting Tyr445 to Thr439. Breaking of either interaction triggers slow inactivation by means of a local disruption in the selectivity filter, while severing the Tyr445-Thr439 H-bond is likely to communicate this conformational change to the adjacent subunit(s). DOI: http://dx.doi.org/10.7554/eLife.01289.001.