Developing and adult forebrains contain neural stem cells (NSCs) but no marker is available to highly purify them. When analysed by flow cytometry, stem cells from various tissues are enriched in a 'side population' (SP) characterized by the exclusion of the fluorescent dye Hoechst 33342. Here, we characterize the SP in embryonic, neonatal and adult forebrains, as well as in neurosphere cultures and we have determined whether this SP could be a source of enriched NSCs. By using specific inhibitors, we found that the SP from embryonic forebrain results from the activity of the ABCG2 transporter, a characteristic of other stem cells, whereas the SP from adult forebrain probably results from the ABCB1 transporter. SP cells from embryonic and adult forebrains, however, expressed a range of cell surface markers more consistent with a haematopoietic/endothelial origin than with a neural origin; NSC markers were mostly expressed on cells outside the SP (in the main population, MP). Moreover, assays for NSC growth in vitro showed that SP cells from embryonic and adult forebrains did not generate NSC-derived colonies, whereas the MP did. We thus conclude that NSCs from developing and adult forebrains are not contained in the SP contrary to stem cells from other tissues.