Single barnacle muscle fibers from Megabalanus Psittacus (Darwin) were internally perfused with a number of solutions of K-acetate of concentration 6"2 which was increased from 100 to 600 mM maintaining a pH of 7.5. The external solutions at the same pH contained 40 mM MgAc 2 and K-acetate of concentration C1 which was varied from 10 through 60 mM. The tonicity of both internal and external fluids was maintained at 1,000 mosmole with sucrose. The membrane potential E m (the potential was referred to the external solution as ground) arising across the fiber membrane was measured using those internal and external solutions which maintained the ratio (C2/C1) at 10. Similar measurements but now maintaining the ratio (C2/C~) at 2, where C~ was the concentration of Tris-C1 in the internal perfusion fluid and was increased from 100 to 250 mM and C2 was the concentration of total C1 in the external fluid which contained 40 mM MgC12 and different amounts of Tris-C1 (120 to 420 raM), were also made using various isotonic solutions at pH 4.0. The measured values of E m corrected for the liquid junction potentials, on interpolation with the theoretical curves of E m plotted against log (1/C1) derived from the theory of membrane potential developed by Teorell and by Meyer and Sievers, gave values of 0.019 and 0.7 M for the density of fixed charges present on the membrane at pH 7.5 (cation selective) and 4.0 (anion selective), respectively. Also, values of 2.3 at pH 7.5 and 1.0 at pH 4.0 were derived for the mobility ratio of cation to anion.It is well recognized that charges present on the surface of biological membranes influence various aspects of biological behavior. A surface charge model has been used by Gilbert and Ehrenstein (1969) to explain some of the voltage-dependent characteristics of the conductance of the nerve fiber. Other biological phenomena, e.g. muscle contraction, protoplasmic streaming, etc., are likely to be greatly influenced by the nature of the charged membrane surfaces. Consequently, a number of techniques, mostly electrokinetic in nature, have been utilized by a number of investigators (see Gilbert, 1971 for a brief review) to estimate the density of charges present in membranes of various biological preparations. Recently,