Using human intermediate-conductance calcium-activated potassium (hIKCa1) channels as a model we aimed to characterize structural differences between maurotoxin (MTX) and charybdotoxin (CTX) and to gain new insights into the molecular determinants that define the interaction of these pore-blocking peptides with hIKCa1 channel. We report here that the block of MTX, but not of CTX on current through hIKCa1 channels is pH0 dependent. The replacement of histidine 236 from hIKCa1 channel with a smaller amino acid, cystein, did not change MTX binding affinity, however, partially affected the pH0 dependency of its block at low pH0. In contrast, CTX binding affinity to the hIKCa1_H236C channel mutant was increased suggesting that His236 might play a role in the binding of CTX, but has only a weak influence in the binding of MTX to hIKCa1 channels.