We have recently published a new technique for visualizing nuclei in living muscle fibers of intact animals, based on microinjection of labeled DNA into single myofibers, excluding satellite cells (Bruusgaard JC, Liestol K, Ekmark M, Kollstad K, and Gundersen K. J Physiol 551: 467–478, 2003). In the present study, we use this technique to study fiber segments of soleus and extensor digitorum longus (EDL) muscles from mice aged 2, 14, and 23 mo. As the animals maturing from 2 to 14 mo, they displayed an increase in size and number of nuclei. Soleus showed little change in nuclear domain size, whereas this increased by 88% in the EDL. For 14-mo-old animals, no significant correlation between fiber size and nuclear number was observed (R² = 0.18, P = 0.51) despite a fourfold variation in cytoplasmic volume. This suggests that size and nuclear number is uncoupled in middle-aged mice. When animals aged from 14 to 23 mo, EDL IIb, but not soleus, fibers atrophied by 41%. Both EDL and soleus displayed a reduction in number of nuclei: 20 and 16%, respectively. A positive correlation between number of nuclei and size was observed at 2 mo, and this reappeared in old mice. The atrophy in IIb fibers at old age was accompanied by a disturbance in the orderly positioning of nuclei that is so prominent in glycolytic fibers at younger age. In old animals, changes in nuclear shape and in the peri- and internuclear microtubule network were also observed. Thus changes in myonuclear number and distribution, perhaps related to alterations in the microtubular network, may underlie some of the adverse consequences of aging on skeletal muscle size and function.