This study aimed to analyse the sarcolemma of Col6a1-/- fibers in comparison with wild type and mdx fibers, taken as positive control in view of the known structural and functional alterations of their membranes. Structural and mechanical properties were studied in single muscle fibers prepared from FDB muscle using atomic force microscopy (AFM) and conventional electrophysiological techniques to measure ionic conductance and capacitance. While the sarcolemma topography was preserved in both types of dystrophic fibers, membrane elasticity was significantly reduced in Col6a1-/- and increased in mdx fibers. In the membrane of Col6a1-/- fibers ionic conductance was increased likely due to an increased leakage, whereas capacitance was reduced, and the action potential (ap) depolarization rate was reduced. The picture emerging from experiments on fibers in culture was consistent with that obtained on intact freshly dissected muscle. Mdx fibers in culture showed a reduction of both membrane conductance and capacitance. In contrast, in mdx intact FDB muscle resting conductance was increased while resting potential and ap depolarization rate were reduced, likely indicating the presence of a consistent population of severely altered fibers which disappear during the culture preparation.