Endurance training represents one extreme in the continuum of skeletal muscle plasticity. The molecular signals elicited in response to acute and chronic exercise and the integration of multiple intracellular pathways are incompletely understood. We determined the effect of 10 days of intensified cycle training on signal transduction in nine inactive males in response to a 1-h acute bout of cycling at the same absolute workload (164 +/- 9 W). Muscle biopsies were taken at rest and immediately and 3 h after the acute exercise. The metabolic signaling pathways, including AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR), demonstrated divergent regulation by exercise after training. AMPK phosphorylation increased in response to exercise ( approximately 16-fold; P < 0.05), which was abrogated posttraining (P < 0.01). In contrast, mTOR phosphorylation increased in response to exercise ( approximately 2-fold; P < 0.01), which was augmented posttraining (P < 0.01) in the presence of increased mTOR expression (P < 0.05). Exercise elicited divergent effects on mitogen-activated protein kinase (MAPK) pathways after training, with exercise-induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation being abolished (P < 0.01) and p38 MAPK maintained. Finally, calmodulin kinase II (CaMKII) exercise-induced phosphorylation and activity were maintained (P < 0.01), despite increased expression ( approximately 2-fold; P < 0.05). In conclusion, 10 days of intensified endurance training attenuated AMPK, ERK1/2, and mTOR, but not CaMKII and p38 MAPK signaling, highlighting molecular pathways important for rapid functional adaptations and maintenance in response to intensified endurance exercise and training.