Anterior pituitary cells from adult male rats were cultured on a natural extracellular matrix (ECM). From the 5th day, spindle-shaped cells, fusing to form elongated fibers, were observed among the epithelial cells. These fibers later increased in size and number and were identified as striated muscle fibers, based on their multi-nucleation, cross-striation and rhythmic contraction. Striated muscle development was further validated by the appearance of cholinergic-nicotinic receptors, as demonstrated by the binding of 125I-alpha bungarotoxin (a-Btx). The effect of various supplements added to the cultures on a-Btx binding was used as a measure of muscle cell differentiation. ECM and serum were essential for myotube formation. Insulin (or IGF-I) and cortisol synergistically caused a 20-fold increase in myogenesis. FGF and EGF were effective only in the presence of insulin and cortisol. The data suggest that: (a) cells, derived from adult pituitary, differentiate, in primary culture, into striated muscle fibers; (b) the induction of this differentiation is multi-factorial; ECM, serum, insulin and glucocorticoids facilitate this myogenesis.