Mitochondria can sense signals linked to changes in energy demand to affect nuclear gene expression. This retrograde signaling pathway is presumed to be involved in the regulation of myoblast proliferation and differentiation. We have investigated the regulation of mitochondrial biogenesis and production of putative retrograde signaling agents [hydrogen peroxide (H₂O₂) and Ca²⁺] during the cell cycle and the onset of differentiation in L6E9 muscle cells. The biosynthesis of cardiolipin and mitochondrial proteins was mainly achieved in S phase, whereas the expression of mitochondrial biogenesis factors [peroxysome proliferator activated receptor (PPAR) , PPAR and neuronal nitric oxide synthase 1] was regularly increased from G1 to G2M phases. In agreement with the increase in mitochondrial membrane potential, mitochondria in S and G2M phases have significant higher H₂O₂ level when compared to G1 phase. By contrast, the onset of differentiation was characterized by a marked reduction in mitochondrial protein expression and mitochondrial H₂O₂ level. The capacity of mitochondria to release Ca²⁺ in response to a metabolic challenge was significantly decreased at the onset of differentiation. Finally, an increase in calmodulin expression in S and G2M phases and a transitory increase in phosphorylated nuclear factor of activated T cells (NFAT) c3 in S phase was observed. NFATc3 phosphorylation was markedly decreased at the onset of differentiation. Our data point to functional links between the control of mitochondrial biogenesis and the regulation of the level of retrograde signaling agents during the cell cycle and the onset of differentiation in L6E9 muscle cells.