The effect of palmitic acid (PA) and oleic acid (OA) on electrical parameters of planar membranes was studied. We found a substantial difference between the effects of PA and OA on proton transfer. PA induced a small increase in conductance, requiring a new technique for estimating proton-mediated currents across low-conductance planar bilayers in which an electrometer is used to measure the transmembrane current under virtual short circuit (SCC). Open-circuit voltage and SCC were used to determine proton and leak conductances. OA caused a marked increase in membrane conductance, allowing the use of a voltage-clamp technique. From SCC data, we were able to estimate the flip-flop rate constants for palmitate (1 x 10(-6) s(-1)) and oleate (49 x 10(-6) s(-1)) anions. Cholesterol, included in the membrane-forming solution, decreased importantly the leak conductance both in membranes unmodified by FA and in membranes modified by PA added to the bath.