The activity of the E2 F-family of transcription factors is tightly linked to control of the cell cycle. p107 and p130, two closely related members of the retinoblastoma protein-family of negative cell cycle regulators, modulate the activity of the E2f-family proteins by direct interaction with these factors. To understand the role of p107 and p130 in progression through or exit from the cell cycle, we have characterized the expression, phosphorylation state, cyclin-binding, and E2f-binding activity of p107 and p130 during terminal differentiation of rat myoblast cells into immature skeletal muscle (myotubes). In exponentially growing L6 myoblasts, p107 is phosphorylated in a cell cycle-dependent manner, and E2f-site binding complexes containing p107 is phosphorylated in a cell cycle-dependent manner, and E2f-site binding complexes containing p107 can be observed throughout the cell cycle. During differentiation of L6 cells, p107 levels are reduced, while p130 protein levels are increased 8-fold. Despite both p107 and p130 becoming hypophosphorylated during myogenesis, the E2F-site DNA-binding complexes containing p107 observed in exponentially growing myoblasts are quantitatively replaced in myotubes with complexes containing only p130. In myotubes, p107 is not associated with E2f-family proteins that are capable of binding DNA. The failure to observe p107-containing complexes in myotubes appears to be due to the differentiation-specific induction of both p130 and cyclin D3, p107 is found in complexes with cyclin D3 in myotubes, and the addition of exogenous cyclin D3 or p130 to lysates from undifferentiated L6 cells was able to disrupt p107-containing E2F-site binding complexes. In myotubes, p130 also forms complexes with cyclin D3 as well as cyclin E, cdk2, and cdk4. We are able to copurify cyclin D3 with cyclin E from myotubes, indicating the presence of a macromolecular complex containing both cyclin E and cyclin D3 simultaneously bound to p130. Thus, in myoblasts, p107 is normally involved in regulation of E2f-family proteins during cell cycle progression, while p130 is a differentiation-specific regulator of E2f activity. Our results also provide evidence that the apparent positive regulator of cell cycle progression, cyclin D3, has a function in terminally differentiated muscle cells.