The distribution of K and Cl permeability, PK and PCl, over the surface and T-tubule membranes of red rat sternomastoid fibers has been determined. Membrane potential, Vm, was recorded with 3-M KCl-filled glass microelectrodes. Changes in Vm with changes in [K]o or [Cl]o were used to estimate PCl/PK in normal and detubulated preparations. The results show that the T-tubule membrane has a high PCl and is therefore different from the T-tubule membrane of amphibian fibers. Analysis of the time course of depolarization when [K]o was raised (in SO4 solutions) showed that PK was distributed over the surface and T-tubule membranes. Two observations suggested that T-tubule PCl was higher than the surface PCl. Firstly, in normal fibers, the depolarization caused by an increase in [K]o was 3.5 times greater in SO4 solutions than in Cl solutions. In marked contrast, the depolarization in glycerol-treated fibers was independent of [Cl]o. Secondly, the rapid change in Vm when [Cl]o was changed was reduced by 80% after glycerol treatment. Both observations suggest that PCl was low in glycerol-treated fibers. PCl/PK was calculated from the Vm data using Goldman, Hodgkin and Katz equations for Na and K or for Na, K, and Cl. In normal fibers PCl/PK = 4.5 and in glycerol-treated fibers PCl/PK = 0.28. Since it is unlikely that glycerol treatment would increase PK, the reduction in the ratio must follow the loss of Cl permeability "channels" in the T-tubule membrane.