Matrix metalloproteinases (MMPs) are believed to be involved in the invasion and metastasis of various human carcinomas. In the present study, the production levels of seven different MMPs (MMP-1, -2, -3, -7, -8, -9, and -13), the activation of the zymogen of MMP-2 (proMMP-2), the expression of membrane-type MMPs (MT1-, MT2-, and MT3-MMPs), and the tissue localization of the activated enzyme were examined in human invasive papillary thyroid carcinomas. Sandwich enzyme immunoassays revealed that among the MMPs examined, only the MMP-2 production level is significantly enhanced in the carcinoma tissues compared with the follicular adenoma and normal control thyroid tissues. Gelatin zymography indicated that the proMMP-2 activation ratio is considerably higher in carcinomas with lymph node metastasis than it is in those without metastasis, follicular adenomas, or normal controls (P < 0.01). Northern blot analysis of the expression of MT1-, MT2-, and MT3-MMPs, which are known to activate proMMP-2 in vitro, demonstrated the predominant expression of MT1-MMP mRNA in the carcinoma tissues (15 of 15 cases), whereas MT2-MMP expression was confined to 26% of the cases (4 of 15 cases), and no consistent expression of MT3-MMP was observed. MT1-MMP mRNA expression levels correlated with the proMMP-2 activation ratio (r = 0.692; P < 0.01), but such a correlation was not obtained with MT2-MMP. There was also a direct correlation between MT1-MMP expression and lymph node metastasis (P < 0.05). In situ hybridization indicated that both carcinoma and stromal cells express MT1-MMP transcripts (five of six cases). MT1-MMP was also immunolocalized to carcinoma and stromal cells in all of the carcinoma samples (26 of 26 cases), which were positive for MMP-2. In situ zymography indicated definite gelatinolytic activity in the carcinoma cell nests, which was abolished by incubation of the carcinoma samples with a synthetic MMP inhibitor before the reaction. These results suggest for the first time that among seven different MMPs, the production of proMMP-2 and its MT1-MMP-mediated activation in the carcinoma cell nests play an important role in the lymph node metastasis of human invasive papillary thyroid carcinomas.