Endurance exercise induces increases in mitochondria and the GLUT4 isoform of the glucose transporter in muscle. Although little is known about the mechanisms underlying these adaptations, new information has accumulated regarding how mitochondrial biogenesis and GLUT4 expression are regulated. This includes the findings that the transcriptional coactivator PGC-1 promotes mitochondrial biogenesis and that NRF-1 and NRF-2 act as transcriptional activators of genes encoding mitochondrial enzymes. We tested the hypothesis that increases in PGC-1, NRF-1, and NRF-2 are involved in the initial adaptive response of muscle to exercise. Five daily bouts of swimming induced increases in mitochondrial enzymes and GLUT4 in skeletal muscle in rats. One exercise bout resulted in approximately twofold increases in full-length muscle PGC-1 mRNA and PGC-1 protein, which were evident 18 h after exercise. A smaller form of PGC-1 increased after exercise. The exercise induced increases in muscle NRF-1 and NRF-2 that were evident 12 to 18 h after one exercise bout. These findings suggest that increases in PGC-1, NRF-1, and NRF-2 represent key regulatory components of the stimulation of mitochondrial biogenesis by exercise and that PGC-1 mediates the coordinated increases in GLUT4 and mitochondria.