In the present study the effect of exercise training was investigated on the expression and activity of matrix metalloproteinases (MMPs) in the human skeletal muscle 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 five weeks. Biopsies were taken from the vastus lateralis muscles of both legs at rest before the training period, after 10 d and five weeks of training, and 2 h after the first exercise bout for analysis of MMP and TIMP-1 mRNA, enzyme activity and protein expression. Levels of MMP-2, MMP-14, and TIMP-1 mRNA in muscle tissue increased after 10 d of training regardless of blood flow condition. MMP-2 mRNA level in laser-dissected myofibers and MMP-2 activity in hole muscle increased with training. The level of MMP-9 mRNA and activity increased after the first bout of exercise. Although the number of transcripts of MMP-2 was significantly greater than that of MMP-9, the activity of MMP-9 after a single bout of exercise was similar to that of MMP-2 after 10 d 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.