In the present study, the effect of exercise training on the expression and activity of matrix metalloproteinases (MMPs) in the human skeletal muscle was investigated. Ten subjects exercised one leg for 45 min with restricted blood flow and then exercised the other leg at the same absolute workload with unrestricted blood flow. The exercises were conducted four times per week for 5 wk. Biopsies were taken from the vastus lateralis muscles of both legs at rest before the training period, after 10 days and 5 wk of training, and 2 h after the first exercise bout for analysis of MMP and tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA, enzyme activity, and protein expression. Levels of MMP-2, MMP-14, and TIMP-1 mRNA in muscle tissue increased after 10 days of training regardless of blood flow condition. MMP-2 mRNA level in laser-dissected myofibers and MMP-2 activity in whole muscle increased with training. The level of MMP-9 mRNA and activity increased after the first bout of exercise. Although MMP-9 mRNA levels appeared to be very low, the activity of MMP-9 after a single bout of exercise was similar to that of MMP-2 after 10 days of exercise. MMP-2 and MMP-9 protein was both present throughout the extracellular matrix of the muscle, both around fibers and capillaries, but MMP-2 was also present within the skeletal muscle fibers. These results show that MMPs are activated in skeletal muscle in nonpathological conditions such as voluntary exercise. The expression and time pattern indicate differences between the MMPs in regards of production sites as well as in the regulating mechanism.