Calcium/Calmodulin-dependent Protein Kinase II (CaMKII) controls gene expression in multiple tissues(1), but its role in regulating adaptation of skeletal muscle is largely unknown. The aims of this study were to investigate the relation between fibre type-dependent CaMKII activity and cytochrome-c oxidase subunit IV (COXIV) protein expression, and the effects of CaMKII overexpression in vivo on COXIV protein expression in adult skeletal muscle fibres. COXIV was used as a marker for mitochondria. Female 3 month-old Wistar rats (250-300 gram) (n=3) were anesthetised with 2-4% isoflurane through inhalation. Plasmids encoding α/β-CaMKII were injected I.M. into the right m. gastrocnemius medialis (GM) or m. soleus and empty plasmids were injected into the contra-lateral muscle, followed by electroporation. Rats were kept in cages for 8 days afterwards. After experiments, transfected muscles were dissected, snap-frozen in liquid nitrogen and stored at -800C until used for analysis. Non-stimulated GM was sampled separately while rats (n=6) were fully anaesthetized (12.5% urethane, 1.2 ml/100g body weight, injected I.P). Proximal and distal portions of non- stimulated GM, which contains compartmentalized fibres with high or low oxidative capacity (2), were analysed separately. Total/phosphoThr286-CaMKII and COXIV protein content in non-stimulated GM was quantified by Western blotting. Transfected fibres were co-stained for total CaMKII and COXIV or phosphoThr286-CaMKII, and imaged with a confocal microscope. COXIV staining was quantified with ImageJ. Data are presented as mean±S.E.M. (arbitrary units). In non-stimulated muscle, levels of phosphoThr286-CaMKII were higher in the distal part of GM (1.00±0.22 vs. 1.85±0.21; p=0.027, t-test), with no difference in total CaMKII (1.00±0.10 vs. 1.10±0.07; p=0.45, t-test). In contrast, COXIV protein content was lower in the distal compared to the proximal part of GM (1.00±0.19 vs. 0.44±0.14; p = 0.024, t-test). Immunohistochemical staining of transfected muscles showed higher phospho-CaMKII levels in CaMKII-transfected fibres compared to non-transfected fibres after cage activity. This showed that CaMKII activity was increased in transfected fibres in vivo. There was, however, no difference in COXIV staining intensity between non-transfected and CaMKII-transfected fibres in both GM (non-transfected: n=51, transfected: n=54; 1.00±0.036 vs. 0.99±0.034, p=0.912, t-test) and m. soleus (non-transfected: n=35, transfected: n=38; 1.00±0.05 vs. 1.07±0.05; p=0.375, t-test). We therefore conclude that increased CaMKII activity is not sufficient to induce mitochondrial biogenesis in rat skeletal muscle.