Summry— Myf-5 and MyoD are the two muscle regulatory factors expressed from the myoblast stage to maintain the identity and to promote the subsequent differentiation of muscle precursor cells. To get insight into their role we have studied the capacity to proliferate and to differentiate of myf-5 and myoD null myoblasts in primary cultures and in the subsequent passages. Our results indicate that myf-5 null myoblasts differ from wild type (wt) myoblasts in that they undergo precocious differentiation: they become myogenin- and troponin T-positive and fail to incorporate bromodeoxyuridine (BrdU) under culture conditions and at a time when wt cells are not yet differentiated and continue to proliferate. In primary cultures of myoD null cells, up to 60% of the cells were scored as myoblasts on the basis of the expression of myf-5. These myoD-deficient myoblasts, unlike myoD-expressing cells, were poorly differentiating and displayed a severe growth defect that led to their elimination from the cultures: within a few passages myoblasts were absent from myoD-deficient cultures, which mostly consisted of senescent cells. That a null mutation in either gene reduces the proliferative potential of cultured myoblasts raises the possibility that Myf-5 and MyoD serve proliferation of muscle precursor cells.