We found a tumor me&stasis-associa ted heparan sulfate (HS)degrading endoglycosidase in melanoma cells that is a unique endo-&ghzuronidase (hepamnam ) capable of speciftcally cleaving HS at intrachain sites (M. Nakajima, T. Irimura, N. DiFerrame, and G. L. Nicolson, 1984, J. Biof. Chem. 259.2283-2290). To perform rapid and microscale quantitative assays of heparanase we developed a solid-phase HS substrate by crosslinking radiolabeled HS onto agarose gel beads using one covalent linkage. The HS from bovine lung was partially Ndesulfated and labeled with ["C]acetic anhydride. Free HS amino groups were completely acetylated, and reducing terminal saccharides were reductively aminated. The HS derivatives with amino groups at their reducing termini were coupled to amino-reactive agarose beads Incubation of the solid-phase HS substrates with B 16 melanoma cell extracts in the presence of ~saccharic acid 1 +lactone (a potent exo-j3glucuronidase inhibitor) resulted in the time-and dosedependent release of [%]HS fragments. Human melanoma cell lines were tested for HS-degrading endoglycosidase using the newly de veloped solid-phase HS substrates. The human malignant melanoma cells tested had high levels of HSdegrading activity that were comparable to those of highly metastatic murine B16-FlO melanoma cells. 0 1986 AC&& Pms, Inc.