Matrix metalloproteinases (MMPs) play a key role in events related to tumor cell invasion and growth. Therefore, we examined tumor tissue samples from 12 individuals with invasive breast cancer for the expression and localization of MMP-1, -2, -3 and -9. These were detected by immunohistochemistry. Simultaneously in this material the matrix-degrading enzyme activity was tested by in situ zymography. Additionally, we used a spheroid model of two breast cancer cell lines (MCF-7 and MDA-MB-435) for MMP-expression and activity by immunohistochemistry and in situ zymography. Semi-quantitative investigation of various MMPs by immunohistochemistry in tissue samples revealed higher expression levels at the tumor periphery where tumor cells grow less compact when compared to compact tumor cell complexes as in the center of the tumors. In situ gelatinolytic activity paralleled these observations showing pronounced activity at the tumor periphery when compared to tumor centers. Similarly, tumor cell spheroids of two cell lines had higher gelatinolytic activity in less densely growing tumor cell groups than in the more densely growing ones. Our study provides considerable evidence that expression and activation of major MMPs is dependent on tumor cell density. This has a major impact on the biological behavior of tumor cells such as invasion and metastasis.