Expression of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2 was studied in non-small-cell lung cancer (NSCLC). Activity of MMP-2 and MMP-9 by gelatin zymography and expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNAs were examined in 11 lung cancer cell lines which included six small-cell lung cancer (SCLC) cell lines. Localization of MMP-2, MMP-9, TIMP-1, and TIMP-2 was examined by immunohistochemistry in 43 resected NSCLC (22 adenocarcinomas, 17 squamous cell carcinomas, 4 large cell carcinomas) using specific anti-human monoclonal antibodies. Expression of MMP-2 mRNA was detected in 5 (100%), MMP-9 in 1 (20%), TIMP-1 in 4 (80%), and TIMP-2 in 5 (100%) of 5 NSCLC cell lines examined. MMP-2 gelatinolytic activity also was detected in all five NSCLC cell lines, whereas MMP-9 activity was detected in only one cell line. In 43 patients, MMP-2, MMP-9, TIMP-1, and TIMP-2 immunoreactivity was demonstrated in 19 (44%), 9 (21%), 15 (35%), and 29 (67%) excised tumors, respectively. All stromal fibroblasts in tumor samples stained positive for MMP-2. There was a correlation between TIMP-2 immunoreactivity and disease stage (42% stage I versus 88% stages II, III, and IV) (p = 0.0024). Both cancer cell lines and NSCLC tumor samples frequently expressed MMP-2, MMP-9, TIMP-1, and TIMP-2; MMP-2 in particular was highly expressed in malignant cells and surrounding fibroblasts. These findings suggest that MMP-2 plays a more important role in invasion of NSCLC than MMP-9 and that TIMP-2 may have clinical relevance in NSCLC.