Skeletal muscle may develop adaptive molecular chaperone enhancements as potentially a defense system through repeated daily exercise stimulation. The present study investigates whether prolonged exercise training alters the expression of molecular chaperone proteins for the long term in skeletal muscle. Mature male Wistar rats were subjected for 8 weeks to either single bout of acute intermittent treadmill running (30 m/min, 5 minx4 times, 5° grade) or prolonged treadmill running training (15-40 m/min, 5 minx4 times, 5°-7° grade). Levels of five molecular chaperone proteins (HSP25, HSP60, GRP78, HSP70, and HSC70) were measured in response to acute exercise and prolonged training. HSP70 levels were increased 6 and 24 h after acute exercise, but expression returned to control level within 2 days. In contrast, prolonged training had a long-term effect on HSP70 expression. Levels of HSP70 were notably increased by 4.5 fold over control 2 days after prolonged training; the enhancement was maintained for at least 14 days after training ended. However, other molecular chaperone proteins did not show adaptive changes in response to prolonged training. In addition, HSP70 enhancement by prolonged exercise training was not accompanied by transcription of HSP70 mRNA. These findings demonstrate that prolonged training can induce long-term enhancement of HSP70 expression without change at the mRNA level in skeletal muscle.