Pathways involved in mitochondrial biogenesis associated with myogenic differentiation are poorly defined. Therefore, CC myoblasts were differentiated into multi-nucleated myotubes and parameters/regulators of mitochondrial biogenesis were investigated. Mitochondrial respiration, citrate synthase- and β-hydroxyacyl-CoA dehydrogenase activity as well as protein content of complexes I, II, III and V of the mitochondrial respiratory chain increased 4-8 fold during differentiation. Additionally, an increase in the ratio of myosin heavy chain (MyHC) slow MyHC fast protein content was observed. PPAR transcriptional activity and transcript levels of PPAR-α, the PPAR co-activator PGC-1α, mitochondrial transcription factor A and nuclear respiratory factor 1 increased during differentiation while expression levels of PPAR-γ decreased. In conclusion, expression and activity levels of genes known for their regulatory role in skeletal muscle oxidative capabilities parallel increases in oxidative parameters during the myogenic program. In particular, PGC-1α and PPAR-α may be involved in the regulation of mitochondrial biogenesis during myogenesis.