Low oxygen tension can influence tumor progression by enhancing angiogenesis, a process that may involve Rho GTPases whose activities have been implicated in tumorigenesis and metastasis. In the present study, we show that hypoxia can increase the mRNA levels and intracellular activities of Rac1 and Cdc42 in a time-dependent manner. The hypoxia-stimulated activities of Rac1 and Cdc42 could be blocked by the phosphatidylinositol 3′-kinase (PI3K) inhibitor LY294002 and the protein tyrosine kinase (PTK) inhibitor genistein but were not affected by the p38MAPK inhibitor SB203580 or the MEK-1 inhibitor PD98059, suggesting that the hypoxia-mediated signals were through PI3K and PTK. Correlating with the increased activities of Rac1 and Cdc42, the expression of the pro-angiogenesis factors HIF-1α and vascular endothelial growth factor (VEGF) was upregulated by hypoxia, whereas the expression of the tumor suppressors von Hippel-Lindau and p53 was down-regulated. Dominant negative N17Rac1 and N17Cdc42 could upregulate the expression of p53 and pVHL but downregulate that of HIF-1α and VEGF under hypoxia. Furthermore, the preconditioned medium from N17Rac1 or N17Cdc42-expressing gastric cancer cells was able to inhibit the proliferation of HUVECs. Our results indicate that PI3K and PTK-mediated activations of Rac1 and Cdc42 are involved in the hypoxia-induced production of angiogenesis-promoting factors and tumor suppressors, and suggest that the Rho family GTPases Rac1 and Cdc42 may contribute to the hypoxia-mediated angiogenesis. © 2006 Wiley-Liss, Inc.