Metalloproteinases have been implicated as important factors mediating the tissue migration of a variety of normal and transformed cells. The conditioned medium (CM) of fetal human astrocytes and five glioma cell lines did not degrade azocoll in suspension, but several proteolytic activities, inhibitable by 1,10-phenanthroline, were detected on sodium dodecyl sulfate-polyacrylamide gels containing gelatin. Both cell types secreted three major proteolytic species (Mr 65,000, 57,000, and 52,000). Two of the glioma lines secreted an additional proteinase (Mr 92,000). After treatment with 12-O-tetradecanoylphorbol-13-acetate, the secretion of the Mr 92,000, 57,000, and 52,000 proteinases was induced or enhanced in all of the cells. The Mr 92,000 and 65,000 proteinases bound specifically to a gelatin affinity column. When purified by preparative gel electrophoresis, the Mr 65,000 proteinase was found to degrade type IV procollagen. The Mr 57,000 and 52,000 species were precipitated by anticollagenase IgG. Tissue inhibitor of metalloproteinases was detected in the CM of all of the cells by substrate gel analysis and immunoprecipitation of [35S]methionine-labeled proteins with anti-tissue inhibitor of metalloproteinases IgG. The glioma lines also secreted various amounts of two smaller inhibitors of metalloproteinases (IMPs), also seen in rabbit brain capillary endothelial cell CM (IMP-1 at Mr 22,000 and IMP-2 at Mr 19,000), and an inhibitor not previously identified (IMP-3 at Mr 16,500). 12-O-Tetradecanoylphorbol-13-acetate stimulated the secretion of tissue inhibitor of metalloproteinases in all of the cells and induced IMPs in some of the glioma lines. When gel filtration chromatography of concentrated CM was used to resolve inhibitors from proteinases, the isolated proteinases had activity against azocoll and the glycoprotein and collagen components of an in vitro model of the extracellular matrix. The secretion of a battery of metalloproteinases by astrocytes may be important in facilitating astrocytic migration during development and in pathological conditions such as inflammation or local invasion of astrocytic neoplasms.