It is well known that IGFs-I and -II stimulate both the proliferation and differentiation of myoblasts, but the role of the IGF binding proteins (IGFBPs) during these processes has not been established. In this study we show that IGF-I analogs with greatly reduced affinity for IGFBPs exhibited about a 10-fold increase in potency in stimulating proliferation (as in other cell types), but up to a 100-fold greater potency than native IGF-I in stimulating L6A1c differentiation. Analysis of conditioned media revealed that L6 cells secrete significant levels of IGFBPs that react with antisera to IGFBP-4, -5 and -6. Steady-state levels of IGFBP-4 mRNA were highest in proliferating myoblasts, while IGFBP-5 mRNA could not be detected in myoblasts although its levels were dramatically increased during IGF- or insulin-stimulated differentiation of myoblasts into myotubes. Elevated IGFBP-6 mRNA levels were found in quiescent cells in serum-free medium. IGF-I and IGF-II treatment elevated IGFBP-5 in conditioned media, but longR3IGF-I and insulin, which do not bind to IGFBPs, had smaller effects. This complex regulation of expression of different IGFBPs not only during different stages of muscle growth and differentiation, but also upon stimulation by IGFs or insulin, suggests that the IGFBPs play a specific and significant role in modulating the actions of the IGFs during myogenesis. Journal of Endocrinology (1995) 144, 539–553