SPARC is a secreted glycoprotein that interacts with extracellular matrix (ECM) proteins to promote de-adhesion of cells from the matrix, thereby inducing a biological state conducive to cell migration. We have demonstrated that SPARC is highly expressed in gliomas (grades II-IV) and promotes glioma invasion in vitro. Therefore, the protein itself or its mechanisms of action might become therapeutic targets to arrest glioma invasion. Vitronectin is an ECM protein found in the blood vessel basement membranes and may promote glioma invasion along these structures. It binds to SPARC in vitro. However, it is not known whether SPARC and vitronectin colocalize and/or interact to contribute to brain tumor cell migration in vivo. In this study, we immuno-histochemically determined if the grade I juvenile pilocytic astrocytomas (JPAs) also express SPARC, if vitronectin is expressed in grades I, II, and IV astrocytomas, and if the proteins colocalize in brain tumors in vivo. We performed western blot analyses to determine if different grades of tumors had different intracellular and/or secreted levels of SPARC and vitronectin. We performed migration assays to determine whether vitronectin is a permissive substrate for glioma migration, and whether the extent of migration correlates with the level of secreted SPARC. Our data demonstrated that JPAs expressed SPARC and secreted significantly higher levels than glioblastomas multiforme (GBMs). Vitronectin was absent from well-preserved tumor but present in areas of disrupted tumor, such as degeneration and/or necrosis. SPARC and vitronectin colocalized only in regions of angiogenesis. We observed that the extent of migration on vitronectin inversely correlated with the level of secreted SPARC: the higher the level, the lesser the migration. These data suggest that the outcome of SPARC - ECM interactions may depend on local SPARC concentrations. The high levels of SPARC secreted by the JPAs, paradoxically, may be more prohibitive for migration on vitronectin than the lower levels secreted by the GBMs. This may account, in part, for the lack of JPA invasion into brain tissue along blood vessel membranes.