The degradation of the basement membrane by matrix-metalloproteinase (MMP) and serine protease is a critical point in tumor invasion and metastasis. We measured the activity of MMP-9 from 28 normal, 12 benign and 126 breast cancer tissues using gelatin zymography with an image analysis system. ProMMP-9 was expressed in 17.5% of the cancer patients compared to 2.5% in 40 non-cancerous tissues (p = 0.014). The mature form of MMP-9 (82 kD) was expressed only in T2-T4 stages. During the early phase of breast cancer (DCIS and T1 stage) progression, only production of proMMP-9 increased. However, as the cancer grew or invaded skin (T2-T4), or with lymphovascular permeation, both production and activation of MMP-9 increased. In conclusion, proMMP-9 production was the main cause of increased MMP-9 activity during the early phase, while both production and activation increased in the late phase of breast cancer.