OBJECTIVE: Hyaluronidase (HAse), a degrading enzyme of hyaluronic acid (HA), is highly expressed in patients with malignant glioma. The purpose of this study was to verify whether HAse is related to the invasion of glioma cells. We also investigated if glioma cells with higher mobility in 2-dimensioal (2-D) method have also higher mobility at 3-dimensional (3-D) environment. METHODS: Malignant glioma cell lines (U87MG, U251MG, U343MG-A, and U373MG) were used, and their HAse expressions were evaluated by HA zymography. The migration ability was evaluated by simple scratch technique. The invasiveness of each cell lines was evaluated by Matrigel invasion assay and HA hydrogel invasion assay. In HA hydrogel invasion assay, colonies larger than 150 micrometer were regarded as positive ones and counted. Statistical analysis of migration ability and invasion properties of each cell lines was performed using t-test. RESULTS: In scratch test to examine migration ability of each cell lines, U87MG cells were most motile than others, and U343MG-A least motile. The HAse was expressed in U251MG and U343MG-A cell lines. However, U87MG and U373MG cell lines did not express HAse activity. In Matrigel invasion assay, the cell lines expressing HAse (U251MG and U343MG-A) were more invasive in the presence of HA than HAse deficient cell lines (U87MG and U373MG). In HA hydrogel invasion assay, the HAse-expressing cell lines formed colonies more invasively than HAse-deficient ones. CONCLUSION: Malignant Glioma cells expressing HAse were more invasive than HAse-deficient ones in 3-dimensional environment. Therefore, it might be suggested that invasion of malignant gliomas is suppressed by inhibition of HAse expression or HA secretion. Additionally, the ability of 2-D migration and 3-D invasion might not be always coincident to each other in malignant glioma cells.