Skeletal muscle displays enormous plasticity to respond to contractile activity with muscle from strength- (ST) and endurance-trained (ET) athletes representing diverse states of the adaptation continuum. Training adaptation can be viewed as the accumulation of specific proteins. Hence, the altered gene expression that allows for changes in protein concentration is of major importance for any training adaptation. Accordingly, the aim of the present study was to quantify acute subcellular responses in muscle to habitual and unfamiliar exercise. After 24-h diet/exercise control, 13 male subjects ( 7 ST and 6 ET) performed a random order of either resistance (8 x 5 maximal leg extensions) or endurance exercise (1 h of cycling at 70% peak O-2 uptake). Muscle biopsies were taken from vastus lateralis at rest and 3 h after exercise. Gene expression was analyzed using real-time PCR with changes normalized relative to preexercise values. After cycling exercise, peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (ET similar to 8.5-fold, ST similar to 10- fold, P < 0.001), pyruvate dehydrogenase kinase-4 (PDK-4; ET similar to 26-fold, ST similar to 39-fold), vascular endothelial growth factor (VEGF; ET similar to 4.5-fold, ST similar to 4-fold), and muscle atrophy F-box protein (MAFbx) ( ET similar to 2-fold, ST similar to 0.4-fold) mRNA increased in both groups, whereas MyoD (similar to 3-fold), myogenin (similar to 0.9-fold), and myostatin (similar to 2-fold) mRNA increased in ET but not in ST (P < 0.05). After resistance exercise PDK-4 (similar to 7-fold, P < 0.01) and MyoD (similar to 0.7-fold) increased, whereas MAFbx (similar to 0.7-fold) and myostatin (similar to 0.6-fold) decreased in ET but not in ST. We conclude that prior training history can modify the acute gene responses in skeletal muscle to subsequent exercise.