Differentiation of myoblasts into myotubes is essential for skeletal muscle development and regeneration. Caspase-3 and caspase-9 are required for efficient myoblast differentiation. The caspase-activated endonuclease activity, CAD, and the DNA-damage repair protein XRCC1 have also been shown to be required to complete differentiation. DNA-damage associated with differentiation is accompanied by phosphorylation of Histone 2AX, an event normally catalysed by kinases ATR, ATM or DNA-PK. However, the kinase responsible for phosphorylation during differentiation is not known. Here we show that inhibition of DNA-PK, but not of ATR or ATM, blocked histone phosphorylation during differentiation. We also show that DNA-PK inhibition and siRNA-mediated DNA-PK knockdown blocked cell fusion. These data implicate a new role for DNA-PK in myogenic differentiation.