The present study was conducted to examine whether gene expression of the insulin signaling pathway is activated in response to a single bout of exercise. Adult male rats underwent a single bout of treadmill exercise for 90 min at 22 m/min on a 6°C incline. The animals were sacrificed immediately after exercise or after a predetermined post-exercise recovery period of up to 48 h, and the level of mRNA in the gastrocnemius muscle was measured by the RT-PCR method. Immediately after exercise, mRNA levels of insulin signal transduction intermediates in the gastrocnemius muscle were lower in the exercise group than in the controls. Following this tendency reduction, mRNA levels of insulin receptor, IRS-1, PI 3-kinase, SH-PTP2 and Nck were higher in the exercise group than in the controls. The levels of PI 3-kinase mRNA was significantly higher in the exercise group than in the controls within 6 h after exercise, while levels of mRNA for insulin receptor, IRS-1, SH-PTP2 and Nck were significantly higher in the the exercise group than in the controls at 48 h after exercise. Gene expression of oncogenes was also affected by a single bout of exercise. Levels of c-fos and c-myc mRNA were significantly higher than in the controls immediately after exercise, while the level of c-jun mRNA was lower in the exercise group at 3 h after exercise. Thus, a single bout of exercise affects the gene expression of insulin signal transduction pathway intermediates and oncogenes in rat gastrocnemius muscle. The present findings suggest that an exercise-induced adaptive increase of skeletal muscle insulin sensitivity is a cumulative effect of a single bout of exercise on the gene expression of insulin signal transduction pathway intermediates.