Heparanase is an endo-beta-D-glucuronidase whose enzymatic targets are the glycosaminoglycan chains of heparan sulfate proteoglycans. Elevated levels of heparanase are associated with the metastatic potential of melanoma cells. Treatment of murine and human melanoma cells with the prototypic neurotrophin nerve growth factor (NGF) increases the production of heparanase by melanoma cells. We previously reported that physiologic concentrations of NGF increased in vitro Matrigel invasion of early passage human brain-metastatic 70W melanoma cells but not melanoma cells metastatic to other sites or nonmetastatic melanoma cells. Here we found that treatment of 70W melanoma cells with neurotrophin-3 (NT-3) increased Matrigel invasion, whereas treatment with neurotrophins other than NGF or NT-3 did not influence invasion. Mutants of NGF that do not bind to the neurotrophin receptor p75NTR or other nonneuronal growth factors were not able to enhance the invasion of 70W melanoma cells. When 70W cells were exposed to anti-sense oligonucleotides directed against p75NTR mRNA, there was a reduction in NGF and NT-3 binding, and the neurotrophins failed to enhance Matrigel invasion. To study to properties of heparanase in NT-regulated melanoma-invasive processes, we developed a sensitive heparanase assay consisting of purified [35S]heparan sulfate subpopulations separated by agarose gel electrophoresis. Incubation of 70W cells with NGF or NT-3, but not BDNF, NT-4/5, or mutant NGF, resulted in increased release of heparanase activity that was capable of degrading a subpopulation of heparan sulfate molecules.