We have used the expression of the muscle form of creatine phosphokinase (M-CPK) to assay myogenic differentiation in the cloned muscle cell line BC3Hl. BC3Hl cells express M-CPK when arrested in the G0 portion of the cell cycle. Addition of the anionic form of brain fibroblast growth factor (B-FGF) rapidly represses synthesis of M-CPK with a half-time of 7 h. Even though B-FGF is not mitogenic for the cells, it causes quiescent BC3Hl cells to exit from the G0 portion of the cell cycle, and to accumulate at a new restriction point approximately 4 to 6 h in the G1 portion of the cell cycle. The repression of M-CPK synthesis by B-FGF is reversible upon removal of B-FGF, and cells which have re-initiated expression of M-CPK have also returned to the G0 portion of the cell cycle. The primary control of M-CPK expression by B-FGF appears to be at the level of gene transcription. We conclude that arrest of cells at G0 but not at other positions in the G1 phase of the cell cycle provides permissive conditions for the expression of muscle-specific proteins, and that defined polypeptide growth factors, in this case B-FGF, are important in the control of the expression of muscle-specific proteins.