Expression of neurofilament proteins, NF140K, NF68K, and NF200K, in C2C12 mouse skeletal muscle cells was studied. Immunofluorescence and immunoblot analyses revealed that NF140K was expressed in proliferating C2C12 cells and its localization was similar to desmin, a muscle-specific intermediate filament protein. NF140K became undetectable in C2C12 cells as muscle cell differentiation proceeded. Reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analyses confirmed the expression of NF140K, NF68K, and NF200K in proliferating C2C12 cells. Sequences of the RT-PCR products of NF140K and NF68K were identical to that of authentic mouse NF140K and NF68K, respectively. The NF140K and NF68K mRNA were down-regulated during myogenesis in contrast to the up-regulation of mRNA encoding troponin-T. Furthermore, subcloned C2C12 cells, which express NF140K at a higher level, exhibited retarded myogenesis, i.e., delayed onset of myosin heavy chain synthesis and myoblast fusion. These results suggest that neurofilament proteins may play an inhibitory role in commitment of muscle cell differentiation. The presence of neurofilament proteins in skeletal muscle cells indicates that desmin, vimentin, and neurofilament proteins can be expressed in a single muscle cell.