The dynamic mechanisms underlying the three-dimensional invasive paradigm of C6 astrocytoma cells has been assessed. Spheroids of C6 astrocytoma cells were implanted into three-dimensional collagen type I gels (vitrogen 100) and individual C6 astrocytoma cell invasion monitored. Time-lapsed videomicroscopy was used to assess the dynamic components of cell invasion in three dimensions while scanning and transmission electron microscopy were used to assess matrix architecture and the static aspects of cell invasion. Videomicroscopy outlined an invasion paradigm continuum with repeating phases. A cell surface ruffling phase was followed by invadopodia extension and pull up phases. For some cells the collagen type I matrix extracellular matrix appeared to modify the ability of C6 cells to carry out their invasion paradigm. C6 astrocytoma cells invading a three-dimensional collagen type I matrix utilize a invasion paradigm made up of a number of phases. A better understanding of the dynamic invasion paradigms of malignant glial cells may be useful in the development of effective treatment strategies to prevent or modify malignant glioma invasion.