We have compared several serum free media for the differentiation of C2C12 myoblasts and assessed the extent of differentiation in several ways including as to active tension generation capability C2C12 cells were allowed to differentiate in Dulbecco s modified Eagle's medium (DMEM) containing Ham's F-12 (F-12), AIM V (AIM), 0 2% Ultroser G in DMEM (Ult-G), and 0 I% Sencin in DMEM (Sericin), compared with in DMEM supplemented with 2% horse serum (HS) or 2% calf serum (CS) C2C12 differentiation was assessed as the extent of myotube for mation, glucose metabolism protein expression sarcomere formation, and active tension generation All serum-free media examined were capable of inducing myotube forma tion and the expression of muscle-specific proteins All serum free media except for F 12 gave the sarcomere structure Active tension generation was observed for cells that differentiated in AIM and Ult-G, but the active tension generated by C2C12 cells that differentiated in Ult-G was only similar to 25% in the case of myotubes that formed in HS The addition of Ult-G to the AIM resulted in improvement of the active tension generation capability, the active tension generated being similar to 3 4x compared to that in HS The approach for assessing muscle cell differentiation presented in this study will be suitable for other studies that involve the differentiation of muscle cells Biotechnol Bioeng 2010,107 894-901 (C) 2010 Wiley Periodicals, Inc