The observation that salt-extracted, devitalized cartilage could be penetrated by malignant tumor cells but was nonpermissive to fibroblastic ingrowth led to the postulate that this matrix might be used as a test connective tissue to discriminate in vitro between noninvasive and invasive tumor cell lines. In a novel in vitro system, salt-extracted, bovine articular cartilage was used as a growth surface for defined noninvasive, invasive and metastatic carcinoma cell lines [RBTCC-2,RBTCC-5 and RBTCC-8], derived from chemical carcinogen-induced tumors of the rat urinary bladder. As monitored by thin-section EM, salt-extracted cartilage was readily penetrated by the invasive and metastatic rat bladder carcinoma cell lines. The metastatic cell line could be differentiated from the invasive, nonmetastatic cell line by its greater depth of invasion. In contrast, noninvasive carcinoma cells as well as normal bladder epithelial cells lacked the capacity to erode and penetrate the extracted matrix of the articular cartilage. Using these defined cell lines, salt-extracted cartilage can be used to reproducibly discriminate between carcinomas having different invasive potentials. This assay system may have diagnostic application for the in vitro staging of tumors.