The channel-forming activity of the lipodepsipeptide syringopeptin 25A (SP25A) was investigated at a tethered bilayer lipid membrane (tBLM) with a dioleoylphosphatidylcholine distal leaflet, anchored to a mercury electrode through a hydrophilic tetraethyleneoxy spacer. SP25A was incorporated in the tBLM from different aqueous solutions by recording a series of impedance spectra over a potential range encompassing non-physiological transmembrane potential (Δϕ) values. Once incorporated, SP25A forms stable ion channels over the narrower range of physiological Δϕ values. Ion flow into and out of the spacer, through the lipid bilayer moiety of the tBLM, was monitored by potential step chronocoulometry and cyclic voltammetry at pH 3, 5.4 and 6.8. Potassium ion flow into the hydrophilic spacer along the SP25A channels, during the negative potential scan, proceeds in two stages, except at the higher pH and lower SP25A concentration adopted, where it proceeds in a single stage. In light of the behavior of SP25A single channel currents reported in the literature, the first stage is ascribed to large channels resulting from the aggregation of small ones, while the second more negative stage is associated with the small channels resulting from the disaggregation of the large ones.