Western blot analysis is a widely used method for the semi-quantitative determination of the concentration of specific proteins in a tissue. To control and correct for equal protein loading error, a protein with relatively constant expression in the tissue is normally used as an internal loading control. In most studies the two main proteins commonly used are actin and α-glycerophosphate dehydrogenase (α-GAPDH) (Dittmer and Dittmer, 2006). However, in contrast to other proteins actin is too abundant in skeletal muscle and α-GAPDH is known to vary with fibre type. Therefore, the primary aim of this study was to investigate whether actin, β-tubulin and α-GAPDH are suitable loading controls for differentiating rat skeletal muscles. The experiments were performed using the extensor digitorum longus (edl, a mainly fast-twitch muscle in adult rats) and the soleus (a predominantly slow-twitch muscle in adult rats) muscles of Wistar rats aged between 1 and 90 days. The rats were killed by CO₂ inhalation and the EDL and soleus muscles from both hind limbs were dissected and rapidly snap-frozen in liquid nitrogen. Proteins were then extracted using NP40 cell lysis buffer. Equal amounts of protein (~10 µg per lane) were then resolved in a 10% polyacrylamide/sodium dodecyl sulphate gel. The proteins were then identified using monoclonal antibodies raised against β-tubulin, actin and α-GAPDH and analysed using Scion Image from NIH. The results show that the concentrations of all three proteins increase with age and that this is most rapid between the age of 1 and 14 days. Thereafter, the concentrations of actin and β-tubulin tended to remain relatively constant and were basically similar in the edl and soleus. On the other hand, the concentration of α-GAPDH was always higher in the edl than in the soleus at all of the ages examined. From these results we suggest that actin, β-tubulin and α-GAPDH are not suitable loading controls for skeletal muscles isolated from animals younger than 14 days.