Voltage-gated Kv1.5 channels contribute to the ultra-rapid cardiac K⁺ current, I_Kur, and have been implicated in atrial fibrillation. We used voltage clamp fluorimetry to directly observe the conformational changes associated with Kv1.5 channel gating by attaching a tetramethylrhodamine-5-maleimide (TMRM) fluorescent probe to substituted cysteine residues in the voltage sensor domain (M394C to R400C). We reveal that the fluorescence report of voltage sensor movement in Kv1.5 is uniquely different from that in the archetypical voltage-gated K⁺ channel, Shaker. Whereas the fluorescence report of voltage sensor movement (from TMRM attached at A359C in the S3-S4 linker) in Shaker channels was mono-exponential and occurred with a similar time course (τ=2.4 ± 0.5 ms at +60 mV; n=4) to ionic current activation (τ=4.5 ± 1.0 ms at +60 mV; n=4), the report of Kv1.5 voltage sensor movement reflected complex conformational changes. In these first reports of Kv1 family channel fluorescence, we show that upon depolarization, TMRM at M394C and A397C in the S3-S4 linker of Kv1.5 channels reported a transient rapidly activating fluorescence deflection that was followed by a prominent rapidly decaying component that represented 27 ± 4 % of the total signal and occurred with a τ of 3.7 ± 0.4 ms at +60 mV (n=4). Using 4-aminopyridine and the ILT triple mutation (V407I, I410L and S414T) to dissociate channel opening from voltage sensor movement, we show that the decaying component of fluorescence was associated with channel opening. Furthermore, inhibition of inactivation (by raising external K⁺ from 3 to 99 mM, or the mutation R487V) abolished the decaying component of fluorescence. Interestingly, raising external K⁺ also increased macroscopic conductance and this was reduced by the mutation R487V. These data suggest that the rapidly decaying component of fluorescence reflects channel inactivation. Furthermore, this inactivation may be responsible for the reduced conductance with 3 mM external K⁺ since raising external K⁺ both abolished the rapidly decaying fluorescence component and increased channel conductance. This implies that with physiological K⁺ conditions, a considerable proportion of Kv1.5 channels become rapidly inactivated upon depolarization and that local changes in external K⁺ may act to critically regulate the availability of Kv1.5 channels and therefore the amplitude of I_Kur current in the heart.