In this study, we analysed the inhibitory potencies, blocking characteristics and putative binding sites of three structurally distinct Kv1.5 channel inhibitors on Kv1.5 channels. IC50 values for S9947, MSD-D and ICAGEN-4 were 0.7 mM, 0.5 mM and 1.6 mM, respectively. The Hill coefficients were approximately 2 for MSD-D and ICAGEN-4, but close to 1 for S9947. All three compounds inhibited Kv1.5 channels preferentially in the open state. In contrast to slow on- and off-rates of apparent binding of MSD-D, ICAGEN-4 and S9947 showed fast on-rates. Utilizing Ala-scanning and in silico modeling we suggest binding of the compounds to the central cavity with crucial residues I508 and V512 in the S6-segment. Residue T480 in the selectivity filter seems to be important for ICAGEN-4 and S9947 inhibition but less important for MSD-D binding. As S9947 and ICAGEN-4 show increased block during depolarizing pulses the formation of a tertiary complex including a potassium ion may increasingly stabilize binding of S9947 and ICAGEN-4.