Since the mitochondrial content of cells is mainly regulated by transcriptional mechanisms, we studied cytochrome c as a representative promoter in a culture model of skeletal muscle differentiation. Previous mutagenesis studies had revealed a critical role for CREB1 in the 10fold upregulation of the cytochrome c promoter. In band shift assays, specific protein-DNA complexes had been found in myoblasts and myotubes. Using myotube nuclear extracts, P-CREB antibody leads to a supershift on a truncated cytochrome c promoter containing one CRE element. Total CREB1 protein levels were the same in myoblasts and myotubes, but two bands (probably alternative splice variants) were detected. The ratio of the upper to lower band comparing blasts to tubes showed a shift between the isoforms as well as a shift in phosphorylation state. In order to analyse possible upstream factors known to regulate CREB1, we activated or inhibited several candidate phosphatases or kinases. However none of these interventions changed cytochrome c promoter activity. Also, calcium measured with FURA-2AM was similar in myoblasts and myotubes. In conclusion, CREB1 seems to play a key role for the regulation of mitochondrial genes during muscle differentiation, however the upstream signals are still unknown.