To investigate whether accretion of myonuclei precedes or follows the increase in fibre cross-sectional area and whether this time course is affected by age, left plantaris muscle of 5- and 25-month-old male Wistar rats was overloaded by denervation of its synergists for 1, 2 or 4 weeks. Contralateral plantaris muscle served as control. Myonuclei were counted in haematoxylin-stained cross-sections. While hypertrophy (33% in young adult) became significant after 2 weeks overload (p<0.01), the myonuclear number was increased only at 4 weeks of overload (p<0.001). The time course and magnitude of hypertrophy were similar in young adult and older rats. Older muscles contained 26% more myonuclei per mm fibre length (p=0.001) and had a 10-fold larger proportion of central myonuclei (p<0.001) than young adult muscles. In conclusion, our data indicate that muscle fibre hypertrophy precedes the acquisition of new myonuclei and that the ability to develop hypertrophy is not attenuated or delayed in 25-month-old rat muscle. © 2010 Elsevier GmbH.