Primary human myoblasts (satellite cells), like other human cells, have a limited life spanin vitro.Here we show that expression of the E6E7 early region from human papillomavirus type 16 can greatly extend the life span of both fetal and satellite cell-derived myoblasts and release them from dependence on the growth factors normally necessary for their proliferation. Expression of either the E6 or the E7 gene alone was not sufficient to confer this phenotype, although expression of E7 did delay cellular senescence. The steady-state level of E6E7 transcripts in clonal cultures correlated with proliferative capacity and inversely with the capacity to differentiate into multinuclear myotubes. The expression of E7 alone markedly inhibited cell fusion in both adult and fetal cultures. These effects on myoblast differentiation could be related in part to the level of retinoblastoma protein (pRb), the major cellular target of E7. Terminal differentiation of skeletal myoblasts is associated with permanent withdrawal from the cell cycle; however, continued expression of E6E7 in differentiated myotubes permits reentry of myotube nuclei into S phase in response to growth factor stimulation. These results support a key role for pRb in the acquisition and maintenance of the differentiated state in human skeletal muscle and, in cooperation with p53, in the control of proliferative capacity and response to external growth factors.