The expression of the retinoblastoma (RB) protein has been studied during in vitro muscle differentiation by immunofluorescence staining with three different antibodies against RB protein. Proliferating mononucleate L6 rat myoblasts showed a low level of expression. As cells began to enter a nonreplicating G0 state, the cell population became heterogeneous. Some nonreplicating cells showed a high level of expression. Nuclei at the two ends of myotubes were strongly positive, whereas centrally located nuclei showed low RB expression. Overexpression of the human RB protein in rat L6 myotubes from a Semliki forest virus (SFV)-based, transient expression vector produced a similar picture. Terminally located nuclei expressed human RB at a much higher level than did the centrally located nuclei. The results suggest that individual nuclei with a multinucleated syncytium may undergo position-dependent specialization.