Cecropin A (CecA) and cecropin B (CecB) added to one side of a bilayer formed from equimolar mixtures of DOPS and DOPE, DPhPS and DPhPE, or DOPS, DOPE, and Chol leads to the formation of well-defined and well-reproducible ion channels of different conductance levels while cecropin P1 (CecP1) does not induce pore formation at micromolar concentrations. We found three populations of CecA channels: pores with weak cationic selectivity, pores with weak anionic selectivity, and pores that were nonselective. The dipole modifiers, flavonoids and styryl dyes, were used to modulate the channel-forming activity of CecA and CecB. The mean conductance of single CecA channels is affected by the influence of dipole modifiers on the lipid packing in the membrane. A decrease in the membrane dipole potential is accompanied by a decrease in the steady-state transmembrane current induced by CecA and CecB in cholesterol-free and cholesterol-containing bilayers. The observed changes in the channel-forming activity might be caused by an increase in the energy barrier for the interfacial accumulation of cecropin monomers. This finding indicates that the negative pole of the cecropin dipole is inserted into the membrane.