Side population (SP) cells identified using the Hoechst 33342 fluorescent dye efflux technique overlap with the tumor stem cell fraction that is responsible for tumorigenesis and recurrence in malignant gliomas. Because diffuse invasion of glioma cells represents the main obstacle to successful therapy and underlies recurrence, we examined the existence of an SP fraction in 8 human glioma cell lines and in a sample of primary glioblastoma (GBM) cells and compared the migration potential of SP cells with that of non-SP cells. The SP cells were detected in U373MG (1.5%), U87MG (1.9%), H4 (2.2%), and primary GBM cells (1.5%). The SP cells displayed approximately 2-fold higher expression of ABCG2, the transporter that is mainly responsible for dye efflux. In monolayer and transwell assays, the migration of SP cells was lower than that of non-SP cells. Furthermore, there was a distinct SP in GBM cells selected for slow migration but not in their fast counterparts. The finding that SP cells have a lower migration potential than non-SP cells suggests that cells underlying the initiation and recurrence of gliomas are able to migrate, albeit less so than other glioma cells. These data may also provide an explanation for the clinical observation that most GBM recur in close proximity to the site of the original tumor.