Cultured cell lines have played an integral role in the study of tumor biology since the early 1900's. The purpose of this study is to evaluate colorectal cancer (CRC) cell lines with respect to progenitor tumors and assess whether these cells accurately and reliably represent the cancers from which they are derived. Primary cancer cell lines were derived from fresh CRC tissue. Tumorigenicity of cell lines was assessed by subcutaneous injection of cells into athymic mice and calculation of tumor volume after 3 weeks. DNA ploidy was evaluated by flow cytometry. Invasive ability of the lines was tested with the MATRIGEL™ invasion assay at 24 or 48 hours. Cells were assessed for the presence of Kirsten-Ras (K-Ras), p-53, deleted in colon cancer (DCC), and Src. Protein profiling of cells and tissue was performed by surface enhanced laser desorption/ionization-time of flight/mass spectroscopy. microRNA expression in cell and tumor tissue samples was evaluated by FlexmiR™ MicroRNA Assays. Four cell lines were generated from tumor tissue from patients with CRC and confirmed to be tumorigenic (mean tumor volume 158.46 mm 3). Two cell lines were noted to be diploid; two were aneuploid. All cell lines invaded the MATRIGEL™ starting as early as 24 hours. K-Ras, p53, DCC, and Src expression were markedly different between cell lines and corresponding tissue. Protein profiling yielded weak-to-moderate correlations between cell samples and respective tissues of origin. Weak-to-moderate tau correlations for levels of expression of human microRNAs were found between cells and respective tissue samples for each of the four pairings. Although our primary CRC cell lines vary in their expression of several tumor markers and known microRNAs from their respective progenitor tumor tissue, they do not statistically differ in overall profiles. Characteristics are retained that deem these cell lines appropriate models of disease; however, data acquired through the use of cell culture may not always be a completely reliable representation of tumor activity in vivo.