Substantial evidence indicates that proteolytic degradation of the extracellular matrix is necessary for invasion and metastasis by cancer cells. Our previous work has demonstrated elevated secretion by cultured ovarian adenocarcinoma cells of two gelatinolytic metalloproteinases, a 72-kDa enzyme resembling matrix metalloproteinase 2 (MMP-2) and a 92-kDa enzyme resembling MMP-9 (Moser et al, Int. J. Cancer 56, 552-559, 1994). To assess the potential in vivo relevance of these enzymes, we have examined ovarian carcinoma ascites using gelatin substrate zymography. MMP species identical to those secreted from several well-characterized ovarian adenocarcinoma cell lines were found in the majority of ascites: MMP-2-like gelatinase (23 of 23 cases) and MMP-9-like gelatinase (18 of 23 cases), suggesting a prevalence of these species in the ovarian carcinoma microenvironment and their availability for tumor-associated proteolysis. The contribution of these proteinases to ovarian cancer invasion was further demonstrated by experiments measuring tumor cell-mediated proteolysis of native endothelial cell extracellular matrix (ECM) and tumor cell invasion of reconstituted basement membrane (Matrigel). These data showed that secretion of type IV collagenase activity by a series of independently isolated ovarian adenocarcinoma cell lines correlated well with the ability of these cells to proteolyze the ECM and invade the basement membrane. Furthermore, we have identified and characterized an ovarian carcinoma-associated gelatinase, the 72-kDa MMP found in conditioned media of the DOV 13 cell line, as MMP-2. This enzyme was identical to the previously described MMP-2 from other sources by Western blot, amino terminal sequence, and substrate specificity. Additionally, a large portion of the MMP-2 activity found in DOV 13 conditioned media is active without organomercurial treatment, suggesting that ovarian cancer cells have an endogenous activator of the zymogen. Together, these data suggest that ECM proteolysis mediated by tumor-associated proteinases plays an important role in the invasion and/or metastasis of ovarian carcinoma.