Ion-induced interfacial dynamics of phospholipid monolayers were studied by various electrochemical techniques. The lipid monolayers were constructed by using the mercapto derivatives of natural lipids that were self-assembled directly onto gold electrode surfaces in a tails-down fashion. The supported lipid assemblies appeared to act as rather effective electron-tunneling barriers with K3Fe(CN)6 as the redox probe, despite a relatively low surface coverage and/or a disordered surface structure. Upon the stimulation by alkaline-earth ions, the lipid layers appeared to undergo surface reorganization, exposing part of the electrode surface which resulted in the formation of microscopic mass-transfer lipid channels. The dimensions and/or the number of these channels increased with increasing ion concentrations, and this ion-gate effect appeared to be quite selective, with the most pronounced effects observed among the series of alkaline-earth ions with Ca2+.