The mRNA of the nuclear coactivator peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) increases during prolonged exercise and is influenced by carbohydrate availability. It is unknown if the increases in mRNA reflect the PGC-1α protein or if glycogen stores are an important regulator. Seven male subjects [23 ± 1.3 yr old, maximum oxygen uptake (V̇o2 max) 48.4 ± 0.8 ml·kg−1·min−1] exercised to exhaustion (∼2 h) at 65% V̇o2 max followed by ingestion of either a high-carbohydrate (HC) or low-carbohydrate (LC) diet (7 or 2.9 g·kg−1·day−1, respectively) for 52 h of recovery. Glycogen remained depressed in LC ( P < 0.05) while returning to resting levels by 24 h in HC. PGC-1α mRNA increased both at exhaustion (3-fold) and 2 h later (6.2-fold) ( P < 0.05) but returned to rest levels by 24 h. PGC-1α protein increased ( P < 0.05) 23% at exhaustion and remained elevated for at least 24 h ( P < 0.05). While there was no direct treatment effect (HC vs. LC) for PGC-1α mRNA or protein, there was a linear relationship between the changes in glycogen and those in PGC-1α protein during exercise and recovery ( r = −0.68, P < 0.05). In contrast, PGC-1β did not increase with exercise but rather decreased ( P < 0.05) below rest level at 24 and 52 h, and the decrease was greater ( P < 0.05) in LC. PGC-1α protein content increased in prolonged exercise and remained upregulated for 24 h, but this could not have been predicted by the changes in mRNA. The β-isoform declined rather than increasing, and this was greater when glycogen was not resynthesized to rest levels.