In previous studies we used the cytochrome c promoter as a representative promoter for studying the regulation of mitochondrial biogenesis during muscle differentiation using the skeletal muscle cell line C2F3. The downstream CREB binding site was found to be crucial for promoter upregulation. In band shift assays, specific protein-DNA complexes had been found from nuclear extracts and CREB-1 and P133-CREB-1 antibodies detected that CREB-1 as well as P-CREB-1 interacts in vitro with this CRE element. Chromatin immunoprecipitation experiments (ChIP) verified that CREB-1 and P-CREB-1 bind to the cytochrome c promoter in vivo. Two anti-CREB-1 and anti-P- CREB-1 immunoreactive bands were found and were identified by selective RT-PCR and specific antibodies as CREB-1a and CREB-1[Delta] derived from alternative splicing. In differentiated cells, CREB-1a and P-CREB-1a were predominant. Expression of a dominant-negative A-CREB protein confirms that CREB-1 plays a key role for the regulation of the cytochrome c promoter during muscle differentiation and that a switch between CREB-1a?and CREB-1[Delta] isoforms is involved in this process.