Because optimal overload-induced skeletal muscle hypertrophy requires angiotensin II (Ang II), the purpose of this study was to determine the effects of blocking Ang II production [via angiotensin converting enzyme (ACE) inhibition] on potential mediators of hypertrophy in overloaded skeletal muscle, namely myonuclear addition and fibroblast content. In a 2 x 2 design, adult (200-225 g) female Sprague Dawley rats were placed into one of four groups (n = 8/group): 7-day skeletal muscle overload, sham-operation, 7-day skeletal muscle overload with ACE inhibition, or sham-operation with ACE inhibition. Functional overload of the plantaris and soleus muscles was produced via bilateral surgical ablation of the synergistic gastrocnemius muscle, while ACE inhibition was accomplished by the addition of the ACE inhibitor enalapril maleate to the animals' daily drinking water (0.3 mg/ml). Myonuclear addition and extrasarcolemmal (ES) nuclear proliferation as measured by in vivo 5-bromo-2'-deoxyuridine labeling were significantly (p 0.05) increased by overload in both the slow-twitch soleus and fast-twitch plantaris muscles. Furthermore, ACE inhibition attenuated these overload-induced increases in the soleus muscle but not in the plantaris muscle. However, the effect of ACE inhibition on soleus ES nuclei was not likely due to differences in fibroblast content, because overload elicited significant increases in vimentin-positive area in the soleus and plantaris muscles that were unaffected by ACE inhibition in either muscle. There was no effect of ACE inhibition on any measure in sham-operated muscles. Collectively, these data indicate that Ang II may mediate the satellite cell response to overload in slow-twitch soleus but not fast-twitch plantaris muscles, and that this effect may occur independently of changes in fibroblast content.