Hypoxia-inducible factor 1α (HIF-1α) functions as a transcription factor that is activated by decreased cellular oxygen concentrations to induce expression of a network of genes involved in angiogenesis, erythropoiesis, and glucose homeostasis. Here we demonstrate that two members of the SRC-1/p160 family of transcriptional coactivators harboring histone acetyltransferase activity, SRC-1 and transcription intermediary factor 2 (TIF2), are able to interact with HIF-1α and enhance its transactivation potential in a hypoxia-dependent manner. HIF-1α contains within its C terminus two transactivation domains. The hypoxia-inducible activity of both these domains was enhanced by either SRC-1 or the CREB-binding protein (CBP)/p300 coactivator. Moreover, at limiting concentrations, SRC-1 produced this effect in synergy with CBP. Interestingly, this effect was strongly potentiated by the redox regulatory protein Ref-1, a dual-function protein harboring DNA repair endonuclease and cysteine reducing activities. These data indicate that all three proteins, CBP, SRC-1, and Ref-1, are important components of the hypoxia signaling pathway and have a common function in regulation of HIF-1α function in hypoxic cells. Given the absence of cysteine residues in one of the Ref-1-regulated transactivation domains of HIF-1α, it is thus possible that Ref-1 functions in hypoxic cells by targeting critical steps in the recruitment of the CBP–SRC-1 coactivator complex.