Interleukin-15 (IL-15) is a cytokine which is highly expressed in skeletal muscle, and which stimulates muscle protein accretion in cultured skeletal muscle fibers. Using parental C2 skeletal myoblasts, no significant effects of IL-15 on skeletal muscle differentiation were observed. To test the hypothesis that IL-15 may stimulate skeletal muscle differentiation if the strong differentiation-inducing effects of autocrine insulin-like growth factor (IGF) production were inhibited, a C2 myoblast subline (C2-pBP4) was stably transfected with an expression vector for rat IGF binding protein-4 (IGFBP-4). Differentiation responses to autocrine and exogenous IGFs in C2-BP4 myoblasts were reduced 3- to 4-fold in C2-BP4 cultures compared to C2-pLXSN cultures, a subline transfected with a control plasmid. Addition of IL-15 to C2-pBP4 myoblasts doubled the number of differentiated muscle cells which arose. These findings indicate that IL-15 can stimulate myogenic differentiation in conditions in which the strongly differentiative effects of the IGFs are inhibited. The differentiative activity of IL-15 may be of physiological significance in conditions in which IGF concentrations are low or in which the IGFs are sequestered by binding proteins.