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During development of the heart, mitochondria proliferate within cardiomyocytes. It is unclear whether this is a response to the increasing energy demand or whether it is part of the developmental program. To investigate the role of the electron transport chain (ETC) in this process, we used transgenic murine embryonic stem (ES) cells in which the green fluorescent protein gene is under control of the a‐myosin heavy chain promoter (a‐MHC), allowing easy monitoring of cardiomyocyte differentiation. Spontaneous contraction of these cells within embryoid bodies (EBs) was not affected by inhibition of the ETC, suggesting that early heart cell function is sufficiently supported by anaerobic ATP production. However, heart cell development was completely blocked when adding antimycin A, an inhibitor of ETC complex III, before initiation of differentiation, whereas KCN did not block differentiation, strongly suggesting that …