The side population (SP) phenotype, defined as the reserpine-blockable ability to efflux the nucleic acid dye Hoechst 33342, has been claimed to be enriched for stem cells in several human normal tissues, cancers and cell lines, and thus may be useful for the identification and isolation of cancer stem cells. We demonstrated the presence of SP fractions in all of seven tested gastrointestinal cancer cell lines. Four cell lines were selected (HT29, HGT101, Caco2 and HRA19a1.1) for detailed phenotypic and behavioural analysis with respect to stem cell characteristics. Cell surface marker analysis showed that, contrary to non-SP cells, the SPs entirely lack the expression of CD34. This difference, however, disappeared when the cells were cultured, rendering both populations CD34-positive. Expression of other putative stem cell markers (CD133, CD44, Hes-1, β-catenin, Musashi-1, Oct-4 and CD117) was identical on SP and non-SPs before and after culturing. Sorted SP and non-SP cells were similarly clonogenic in vitro, tumourigenic in vivo, and displayed similar multipotential differentiation potential in vitro and in vivo. Additionally, culturing cytometrically-sorted SP and non-SP cells showed that the populations are interconvertible, each giving rise to the other. Expression of ABCG2 and Mdr-1, two membrane transporter proteins that have been suggested to be responsible for the drug-effluxing capacities of SP cells, including Hoechst 33342, was identical in non-SP and SP cells, indicating that there may be additional factors responsible for the Hoechst effluxing property in gastrointestinal cancer SP cells. Here, we show that the SP and non-SP fractions, albeit phenotypically distinct populations, do not differ with respect to stem cell-like cell number or behaviour. We thus conclude that the concept of the SP phenotype as a universal marker for stem cells does not apply to gastrointestinal cancer cells. These findings stand in contrast to the observations made in many other tissues and harbour important implications for the future search for intestinal cancer stem cell markers. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.