The hypoxic response of mammalian cells is controlled through a transcriptional pathway that is mediated by the hypoxia-inducible factor (HIF). Here, we show that HIF-1 alpha undergoes post-translational modification by the three isoforms of the small ubiquitin-related modifier (SUMO-1, -2 and -3) in vitro in proximity to and within the oxygen-dependent degradation domain (ODDD). SUMO conjugation is promoted in vitro by the E3 SUMO ligase RanBP2/Nup538 and SUMO modification in vivo does not change HIF-1 alpha turnover rate. Using cotransfection of siRNA targeted to endogenous HIF-1a together with HIF-1 alpha siRNA-resistant expression vectors carrying mutations for SUMO modification we demonstrate increased hypoxia-response element-dependent transcriptional activity for SUMO-deficient HIF-1 alpha. These results indicate that when HIF-1 alpha is conjugated to SUMO its transcriptional activity is decreased and that this is not mediated by a change in the protein's half-life. (c) 2007 Elsevier Inc. All rights reserved.