The time course of the intracellular pH (pHi) of barnacle muscle fibers was followed using microelectrodes while the fibers were exposed to 15 mM of the weak acid 5,5-dimethyloxazolidine-2,4-dione (DMO) at pH 6.5. The rapid initial fall in pHi was used to determine the membrane permeability to the DMO molecule, while the much slower fall during the subsequent plateau phase yielded the permeability to the DMO ion. This experimental approach and the mathematical treatment of the data can be used to obtain the membrane permeabilities to other weak acids or bases. We found values of 1.9 X 10(-4) cm/s for the permeability to the DMO molecule and 1.5 X 10(-7) cm/s for the permeability to the ion, assuming the fiber to be a cylinder. Thus the permeability of the neutral form is about 10(3) times that of the ionized form. At commonly encountered conditions of pHi = 7.3, outside pH = 7.5, and membrane potential = -52 mV, this permeability ratio introduces an error of only -0.01 to -0.02 into the determination of pHi based on the distribution of DMO.