Tumor growth is dependent on the ability of neoplastic cells to induce angiogenesis. Remodelling of blood vessels requires reconstruction of the collagen (type IV) and non-fibrous protein components of basement membrane. This study assessed the general protease and collagenase (IV) activities of C6 astrocytoma cells in monolayer and spheroid culture and C6 astrocytoma spheroids growing in vivo. Extracellular release of non-specific proteases and collagenase IV was maximal during early exponential cell growth. Increased spheroid size resulted in enhanced extracellular activity of both enzyme groups assessed. The size of the implanted spheroid influenced the activity measured in vivo. General proteolytic activity was significantly greater in tumor tissue at all spheroid sizes while only the implantation of 750 microns spheroids resulted in significantly increased collagenase type IV activity. The growth of C6 astrocytoma cells in monolayer and spheroid culture in vitro and in vivo is associated with distinct alterations in intracellular and extracellular activity of the proteolytic enzymes assessed. Increased extracellular release of these enzymes may play important roles in tumor-associated angiogenesis, tumor invasiveness, tumor induced hemorrhage and tumor-associated edema.