Previous work has shown that the fractional blockade of the HERG potassium channel by the scorpion toxin BeKm-1 has a negative dependence on voltage, and strong depolarisation of the cell membrane can eliminate this blockade (Milnes et al. 2003; Zhang et al. 2003). These studies arrived at contradictory conclusions regarding whether or not the mechanism of block involves voltage-dependent changes in BeKm-1’s affinity for the channel. Here we used whole-cell patch-clamp at 37°C of HEK 293 mammalian cells expressing heterologous HERG to show that the time course of development (τ) of channel blockade after repolarisation of the membrane varies from 1404 ± 25 ms (mean ± S.E.M.) at 50 nM BeKm-1 to 384.2 ± 0.2 ms at 500 nM (P < 0.001, ANOVA with Bonferroni post-test, n = 6-15); furthermore, at these two concentrations the τ of blockade following repolarisation to 0 mV is identical to the τ of blockade following rapid wash on of BeKm-1 while the membrane is held at 0 mV (not significant, ANOVA with a Bonferroni post-test). We also found that 500 nM of BeKm-1 results in highly potent block (95.00 ± 0.03%, n = 5) when tail currents (-40 mV) were measured after a short (30 ms) depolarisation to +40 mV, although prolonging the depolarizing pulse to 210 ms resulted in significant time-dependent reduction in block by 500 nM BeKm-1 (ANOVA, P <0.001); this is compared with previous results suggesting that maximal blockade of HERG by BeKm-1 does not approach 100% (Zhang et al. 2003). In addition, strong depolarisation (+80 mV for 3 s) can result in an acceleration of toxin wash-off from the channel when held at -80 mV for a further 7 s (P < 0.001, ANOVA with a Bonferroni post-test, n = 6-9 cells). Finally, strong depolarisation can lead to apparent switching from block by Ergtoxin to block by BeKm-1 (as judged by time course) when in the presence of both toxins (P < 0.001, n = 4-6). These data are concordant with voltage-dependent changes in HERG's affinity for the two toxins, and the data diverge from a model in which the toxin is continuously resident in its binding site on the channel in a voltage-independent manner.