The purpose of this study was to determine the effects of functional overload (FO) combined with growth hormone/insulin-like growth factor I (GH/IGF-I) administration on myonuclear number and domain size in rat soleus muscle fibers. Adult female rats underwent bilateral ablation of the plantaris and gastrocnemius muscles and, after 7 days of recovery, were injected three times daily for 14 days with GH/IGF-I (1 mg/kg each; FO + GH/IGF-I group) or saline vehicle (FO group). Intact rats receiving saline vehicle served as controls (Con group). Muscle wet weight was 32% greater in the FO than in the Con group: 162 ± 8 vs. 123 ± 16 mg. Muscle weight in the FO + GH/IGF-I group (196 ± 14 mg) was 59 and 21% larger than in the Con and FO groups, respectively. Mean soleus fiber cross-sectional area of the FO + GH/IGF-I group (2,826 ± 445 μm2) was increased compared with the Con (2,044 ± 108 μm2) and FO (2,267 ± 301 μm2) groups. The difference in fiber size between the FO and Con groups was not significant. Mean myonuclear number increased in FO (187 ± 15 myonuclei/mm) and FO + GH/IGF-I (217 ± 23 myonuclei/mm) rats compared with Con (155 ± 12 myonuclei/mm) rats, although the difference between FO and FO + GH/IGF-I animals was not significant. The mean cytoplasmic volume per myonucleus (myonuclear domain) was similar across groups. These results demonstrate that the larger mean muscle weight and fiber cross-sectional area occurred when FO was combined with GH/IGF-I administration and that myonuclear number increased concomitantly with fiber volume. Thus there appears to be some mechanism(s) that maintains the myonuclear domain when a fiber hypertrophies.