To determine the roles of the retinoblastoma gene (Rb-1) in skeletal muscle differentiation in vitro, we isolated C2 myoblasts stably expressing an antisense RNA directed to the 3'-untranslated region (3'UTR) of Rb-1 mRNA. The levels of Rb-1 mRNA and its product (pRb) in the clones transfected with antisense Rb were markedly decreased to 25-35% of those in the control clone. Cell growth of the clones was accelerated, especially in medium containing low concentrations of fetal calf serum. Even in differentiation medium with a low mitogen level, the antisense Rb clones proliferated as single-nucleated myoblast-like cells without expressing the sarcometric myosin heavy chain protein, whereas the control clone formed highly multinucleated myotubes after 4 days of culture under the same conditions. Under this condition, the levels of Rb-1 mRNA and pRb in the antisense Rb clones were 30-50% of those in the control clone, and no divergent increase in the Rb-family protein p107 expression was observed. This inhibited differentiation was abrogated by reintroducing expression vectors for the sense 3'UTR of Rb-1 mRNA or Rb-1 mRNA lacking its 3'UTR to the clone transfected with antisense Rb. In the antisense Rb clone cultured in differentiation medium, the amounts of MyoD and myogenin mRNA were markedly decreased on the 2nd day of culture in the differentiation medium. The expression of cell cycle-promoting genes including E2F-1 and cyclin D1 was up-regulated throughout the experiment. These results demonstrate that pRb is essential for the completion of terminal differentiation in C2 cells.