Degradation and invasion of basement membrane by tumor cells involves the cooperative hydrolysis of proteoglycans, collagens, and glycoproteins mediated by a number of enzymes including proteases, collagenases, and glycosidases. In order to study these processes in vitro, a tissue culture system was developed in which bovine corneal endothelial cell extracellular matrix (ECM) serves as a substrate for attachment and degradation by human ovarian carcinoma cells. Using this system, a correlation was observed between solubilization of glycoconjugates present in ECM and extracellular levels of beta-N-acetylglucosaminidase (EC 3.2.1.30). To determine the role of individual isoenzymes of beta-N-acetylglucosaminidase (beta-NAG) in ECM degradation, the cellular and secreted forms of the enzyme were fractionated and characterized. Three intracellular isoenzyme forms A, I, and B, were isolated from invasive human ovarian carcinoma cell line A-121. In cell homogenate, forms A and B corresponded to 65 and 33% of total beta-NAG activity, respectively. Form I was found to be localized in the plasma membrane fraction of these cells. Two secreted forms of beta-NAG (As and Bs) were detected in serum-free medium. The separated intracellular and secreted isoenzymes demonstrated similar Km values, ranging from 1 to 5 mM, with p-nitro-B-N-acetylglucosaminide substrate. Treatment of [3H]glucosamine-labeled ECM with the separated isoenzymes of beta-NAG resulted in time- and concentration-dependent releases of radioactivity with potency of 1 greater than B much greater than A. These results suggest that human ovarian carcinoma cell beta-N-acetylglucosaminidase isoenzymes (forms B and A) contribute to ECM degradation as secreted enzymes and form I as a membrane enzyme.