We have examined regulation of the E2F transcription factor during differentiation of muscle cells. E2F regulates many genes involved in growth control and is also the target of regulation by diverse cellular signals, including the RB family of growth suppressors (e.g., the retinoblastoma protein [RB], p107, and p130). The following aspects of E2F function and regulation during muscle differentiation were investigated: (i) protein-protein interactions, (ii) protein levels, (iii) phosphorylation of the E2F protein, and (iv) transcriptional activity. A distinct E2F complex was present in differentiated cells but not in undifferentiated cells. The p130 protein was a prominent component of the E2F complex associated with differentiation. In contrast, in undifferentiated cells, the p107 protein was the prominent component in one of three E2F complexes. In addition, use of a differentiation-defective muscle line provided genetic and biochemical evidence that quiescence and differentiation are separable events. Exclusive formation of the E2F-p130 complex did not occur in this differentiation-defective line; however, E2F complexes diagnostic of quiescence were readily apparent. Thus, sole formation of the E2F-p130 complex is a necessary event in terminal differentiation. Other changes in E2F function and regulation upon differentiation include decreased phosphorylation and increased repression by E2F. These observations suggest that the regulation of E2F function during terminal differentiation may proceed through differential interaction within the RB family and/or phosphorylation.