This investigation examined gingival crevicular fluid (GCF) levels of lysosomal cystein protease, cathepsin B (CAB), during human orthodontic tooth movement. The study included 10 patients (five males, mean age 22.5 +/- 2.8 years and five females, mean age 23.4 +/- 3.9 years), each having one tooth undergoing orthodontic movement, while the contralateral and antagonist teeth were used as the controls. The GCF was sampled at the control and treatment (compression) sites before activation and at 1, 24, and 168 hours. Prevention of plaque-induced inflammation allowed this study to focus on the dynamics of mechanically stimulated CAB levels in GCF. The CAB levels in GCF were determined by fluorospectrometry, using Z-Arg-Arg-MCA as the substrate and by Western blotting analysis. The GCF levels of CAB for the treated teeth were significantly (P< 0.001) higher than those of the control teeth at 24 hours. At the control sites, CAB levels at 24 hours did not change significantly with time. At the experimental site where orthodontic forces were applied, Western blot analysis demonstrated that the molecular forms were 29 kDa mature enzymes. These results indicate that the amount of CAB in GCF is increased by orthodontic tooth movement. This increased CAB may be involved in extracellular matrix degradation in response to mechanical stress.