During transition from rest to exercise, metabolic reaction rates increase sub- stantially to sustain intracellular ATP use. These metabolic demands activate several kinases that initiate signal transduction pathways which modulate tran- scriptional regulation of mitochondrial biogenesis. The purpose of this study was to determine whether metabolic fluctuations per se affect the signaling cascades known to regulate peroxisome proliferator-activated receptor c coac- tivator-1a (PGC-1a). On two separate occasions, nine men performed a con- tinuous (30-min) and an intermittent exercise (30 9 1-min intervals separated by 1-min of recovery) at 70% of V_O2peak. Skeletal muscle biopsies from the vastus lateralis were taken at rest and at +0 h and +3 h after each exercise. Metabolic fluctuations that correspond to exercise-induced variation in metabolic rates were determined by analysis of VO2 responses. During intermittent exercise metabolic fluctuations were 2.8-fold higher despite iden- tical total work done to continuous exercise (317 ` 41 vs. 312 ` 56 kJ after intermittent and continuous exercise, respectively). Increased phosphorylation of AMP-activated protein kinase (AMPK) (~2.9-fold, P < 0.01), calcium/cal- modulin-dependent protein kinase II (CaMKII) (~2.7-fold, P < 0.01) and p38-mitogen-activated protein kinase (MAPK) (~4.2-fold, P < 0.01) occurred immediately in both exercises and to a greater extent after the intermittent exercise (condition x time interaction, P < 0.05). A single bout of intermittent exercise induces a greater activation of these signaling pathways regulating PGC-1a when compared to a single bout of continuous exercise of matched work and intensity. Chronic adaptations to exercise on mitochondria biogene- sis are yet to be investigated.