Cells within a tumor are highly heterogeneous with respect to a wide range of genotypic and phenotypic characteristics. The latter include such properties as growth, survival, invasion, and metastasis. We asked whether the degree to which individual tumor cells rely on a tumor's vasculature might also be heterogeneous. By adapting an intravital Hoechst 33342 staining technique, we labeled and isolated tumor cells based on their relative proximity to perfused vessels. Because tumor regions distal to the vasculature are likely hypoxic, we examined cells deficient for hypoxia-inducible factor-1alpha (HIF-1alpha), a transcription factor that has been shown to mediate hypoxia-induced responses, including apoptosis. Despite reduced vascularization in HIF-1alpha-/- embryonic stem cell-derived tumors, their growth in vivo was found to be accelerated relative to HIF-1alpha+/+ tumor counterparts. We hypothesized that this paradoxical observation is because of decreased apoptotic rate, resulting in diminished vascular dependence of HIF-1alpha-/- cells. Analysis of heterogeneous tumors established from mixtures of HIF-1alpha+/+ with HIF-1alpha-/- cells revealed that the proportion of cells expressing wild-type HIF-1alpha was increased in perivascular areas and decreased in distal tumor regions. Thus, cells expressing HIF-1alpha were found to be highly dependent on proximity to blood vessels for their growth and survival in vivo, whereas cells that had lost HIF-1alpha expression were much less so. Heterogeneity in angiogenesis dependence was also observed among cell subpopulations isolated from human melanoma xenografts. This potential for selection of less vascular-dependent tumor cell variants throughout the course of disease progression may have important implications for the long-term efficacy of anti-angiogenic therapy.