The cellular mechanisms by which contractile activity stimulates skeletal muscle hypertrophy are beginning to be elucidated and appear to include activation of the phosphatidylinositol 3-kinase signaling substrate mammalian target of rapamycin (mTOR). We examined the time course and location of mTOR phosphorylation in response to an acute bout of contractile activity. Rat hindlimb muscle contractile activity was elicited by high-frequency electrical stimulation (HFES) of the sciatic nerve. Plantaris (Pla), tibialis anterior (TA), and soleus (Sol) muscles from stimulated and control limbs were collected immediately or 6 h after stimulation. HFES resulted in mTOR phosphorylation immediately after (3.4 +/- 0.9-fold, P < 0.01) contractile activity in Pla, whereas TA was unchanged compared with controls. mTOR phosphorylation remained elevated in Pla (3.6 +/- 0.6-fold) and increased in TA (4.6 +/- 0.9-fold, P < 0.05) 6 h after HFES. Interestingly, mTOR activation occurred predominantly in fibers expressing type IIa but not type I myosin heavy chain isoform. Furthermore, HFES induced modest ribosomal protein S6 kinase phosphorylation immediately after exercise in Pla (0.4 +/- 0.1-fold, P < 0.05) but not TA and more markedly 6 h after in both Pla and TA (1.4 +/- 0.4-fold vs. 2.4 +/- 0.3-fold, respectively, P < 0.01). Akt/PKB phosphorylation was similar to controls at both time points. These results suggest that mTOR signaling is increased after a single bout of muscle contractile activity. Despite reports that mTOR is activated downstream of Akt/PKB, in this study, HFES induced mTOR signaling independent of Akt/PKB phosphorylation. Fiber type-dependent mTOR phosphorylation may be a molecular basis by which some fiber types are more susceptible to contraction-induced hypertrophy.