The invasive cellular behavior of malignant gliomas is determined by receptor mediated cell-substratum contacts and cell-cell interaction as well as cellular locomotion. This study attempts to break down the complex phenomena of the invasive process into their components of attachment to neighboring cells, aggregate formation, adhesion to matrix substratum, migration and invasion into three-dimensional cellular aggregates separately analyzed in different in vitro assay systems. Using a panel of 13 glioma cell lines, adhesion to non-specifically or merosin coated surfaces was correlated to monolayer cell migration and dissemination of tumor cells from aggregates plated on these substrates. The formation kinetics of aggregates were determined and compared to the ability of these cells to rapidly attach and form mechanically stable cell-cell contacts. The motility rates in the different assay systems as well as cell-cell attachment was correlated to invasion of re-aggregated tumor cells into fetal rat brain. A tight positive correlation was found for substrate adhesion and monolayer migration. In contrast, cell-substratum contacts had little influence on dissemination of cells out of three-dimensional aggregates and no association between monolayer migration and migration of cells out of aggregates was detected. The ability of glioma cells to rapidly form aggregates was associated with enhanced migration out of aggregates. The capacity to invade fetal rat brain aggregates was correlated with the capacity to form stable intercellular adhesion as measured in a cell-cell adhesion assay. Invasion in this system was not found to be associated with migration in monolayer or with migration out of tumor aggregates. This study highlights that current in vitro assays for invasion only represent isolated aspects of the multi-cascade process which is involved in tumor cell invasion.