Peroxisome proliferator-activated receptor gamma coactivator (PGC-1α) is a transcriptional coactivator that plays a key role in coordinating mitochondrial biogenesis. Recent evidence has linked p38 MAPK and AMPK with activation of PGC-1α. It was recently shown in rodent skeletal muscle that acute endurance exercise causes a shift in the subcellular localization of PGC-1α from the cytosol to the nucleus, allowing PGC-1α to coactivate transcription factors and increase mitochondrial gene expression, but human data are limited and equivocal in this regard. Our purpose was to examine p38 MAPK and AMPK activation, and PGC-1α protein content in whole muscle, cytosolic, and nuclear fractions of human skeletal muscle following an acute bout of endurance exercise. Eight trained men (29 ± 3 yr; V̇o2peak = 55 ± 2 ml·kg−1·min−1) cycled for 90 min at ∼65% of V̇o2peak and needle biopsy samples (vastus lateralis) were obtained before and immediately after exercise. At rest, the majority of PGC-1α was detected in cytosolic compared with the nuclear fractions. In response to exercise, nuclear PGC-1α protein increased by 54% ( P < 0.05), yet whole muscle PGC-1α protein was unchanged compared with rest. Whole muscle and cytosolic p38 MAPK phosphorylation increased several-fold immediately after exercise compared with rest ( P < 0.05). Acetyl CoA carboxylase (ACC) phosphorylation, a marker of AMPK activation, was increased by ∼5-fold in cytosolic fractions following exercise ( P < 0.05). These data provide evidence that, in human skeletal muscle, activation of cytosolic p38 MAPK and AMPK may be potential signals that lead to increased nuclear abundance and activation of PGC-1α in response to an acute bout of endurance exercise.