AIM: To characterise differences between three widely used colorectal cancer cell lines using ultrastructural selective staining for glycogen to determine variation in metastatic properties. METHODS: Transmission electron microscopy was used in this investigation to help identify intracellular structures and morphological features which are precursors of tumor invasion. In addition to morphological markers, we used selective staining of glycogen as a marker for neoplastic cellular proliferation and determined whether levels of glycogen change between the three different cell lines. RESULTS: Ultrastructural analysis revealed morphological differences between the cell lines, as well as differentiation into two sub-populations within each cell line. Caco-2 cells contained large glycogen deposits as well as showing the most obvious morphological changes between the two sub-populations. SW480 cells also contained large glycogen stores as well as deep cellular protrusions when grown on porous filter membranes. HT-29 cells had trace amounts of glycogen stores with few cellular projections into the filter pores and no tight junction formation. CONCLUSION: Morphology indicative of metastatic properties coincided with larger glycogen deposits, providing strong evidence for the use of selective staining to determine the neoplastic properties of cells.