To clarify the significance of the balance between matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in the progression of renal cell carcinoma, we transfected both the MMP-2 and TIMP-2 genes simultaneously into RenCa, a mouse renal cell carcinoma cell line that does not express detectable levels of either MMP-2 or TIMP-2 mRNAs, and established several clones with various MMP-2:TIMP-2 expression ratios. On the basis of the quantitative evaluation of the MMP-2: TIMP-2 mRNA expression ratio by Northern blot analysis, we selected a clone overexpressing MMP-2 alone (RenCa/M), a clone overexpressing TIMP-2 alone (RenCa/T), and two kinds of clones overexpressing both, i.e., one with a high (RenCa/MTh) and one with a low (RenCa/MTl) MMP-2: TIMP-2 ratio, to compare the tumor cell phenotypes. In an in vitro tumor cell invasion assay, the MMP-2:TIMP-2 ratios of the RenCa sublines were directly correlated with their invasive potential. The invasive abilities of the parental RenCa cells induced by conditioned media from RenCa sublines were also correlated with the MMP-2:TIMP-2 ratios of the sublines. The cell adhesion assay showed the inverse correlation between the MMP-2 expression levels in the sublines and their cell adhesion to several extracellular matrix components. Furthermore, when injected i.v. or into the renal subcapsule in syngeneic mice, RenCa sublines formed metastatic nodules in the lungs, and the number of nodules was correlated with the MMP-2:TIMP-2 ratio of each clone. In contrast, despite the growth-inhibitory effects of TIMP-2 overexpression, MMP-2 overexpression had no effect on either proliferation in vitro of RenCa sublines or on their growth as tumors in vivo. These results suggest that the MMP-2:TIMP-2 expression ratio is a critical factor in the invasion and metastasis of renal cell carcinoma.