1. Single, fast glycolytic skeletal muscle fibres were isolated from wild‐type (MyoD+/+) and MyoD mutant mice (MyoD‐/‐), which lack a functional copy of the MyoD gene. Fibres were chemically permeabilized to permit manipulation and control of the ionic environment of the otherwise intact myofilament apparatus. 2. Results show a fivefold greater variability in the [Ca2+] required for half‐maximum tension generation among individual MyoD‐/‐ fibres in comparison with controls (p < 0.05). 3. Consistent with this finding, Western blot analysis showed a sevenfold greater variability in the isoform expression pattern of the thin filament regulatory protein troponin T in Myod‐/‐ compared with control fibres (p < 0.05). 4. Electrophoretic analysis of single‐fibre segments indicated no apparent alteration in the isoform expression pattern of other regulatory and contractile proteins. In addition, other parameters of contractile function, including velocity of unloaded shortening, and maximum force production, were not significantly different between MyoD‐/‐ and MyoD ø fibres. 5. These findings indicate that the thin filament structure‐ function relationship is altered due to the MyoD mutation and suggest that MyoD plays a role in establishing and/or maintaining the differentiated phenotype of adult fast skeletal muscle fibres.