Although advances have been made in understanding the role of hypoxia in the stem cell niche, almost nothing is known about a potentially similar role of hypoxia in maintaining the tumor stem cell (TSC) niche. Here we show that a highly tumorigenic fraction of side population (SP) cells is localized in the hypoxic zones of solid tumors in vivo. We first identified a highly migratory, invasive, and tumorigenic fraction of post-hypoxic side population cells (SPm(hox) fraction) in a diverse group of solid tumor cell lines, including neuroblastoma, rhabdomyosarcoma, and small-cell lung carcinoma. To identify the SPm((hox)) fraction, we used an "injured conditioned medium" derived from bone marrow stromal cells treated with hypoxia and oxidative stress. We found that a highly tumorigenic SP fraction migrates to the injured conditioned medium in a Boyden chamber. We show that as few as 100 SPm((hox)) cells form rapidly growing tumors in vivo. In vitro exposure to hypoxia increases the SPm((hox)) fraction significantly. Quantitative real-time polymerase chain reaction and immunofluorescence studies showed that SPm((hox)) cells expressed Oct-4, a "stemness" gene having a potential role in TSC maintenance. In nude mice xenografts, SPm((hox)) cells were localized to the hypoxic zones, as demonstrated after quantum dot labeling. These results suggest that a highly tumorigenic SP fraction migrates to the area of hypoxia; this migration is similar to the migration of normal bone marrow SP fraction to the area of injury/hypoxia. Furthermore, the hypoxic microenvironment may serve as a niche for the highly tumorigenic fraction of SP cells.