MyoD is a murine gene, prototype of a family of genes able to induce muscle differentiation when introduced into a variety of cells. We focused on previously reported, poorly characterized growth-inhibiting properties of MyoD. In a transient expression system, it was found that MyoD can inhibit cell proliferation when expressed in a number of "normal" and transformed murine cell lines. Growth inhibition is not due to induction of differentiation, as demonstrated by means of a variety of approaches. including the expression and evaluation of two series of MyoD mutants. Also, growth arrest due to cell death was excluded. MyoD is able to exert its growth-arresting effects independent of the status of the cell line (whether "normal" or transformed) with the same efficiency. as demonstrated by a series of experiments with NIH/3T3 cells transformed by a number of oncogenes. Although the molecular mechanisms responsible for this effect remain to be elucidated, MyoD appears to be a true growth-arresting gene, at least in the experimental system employed.