Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric DNA-binding complex of the subunits α and β with relevance in O₂ and energy homeostasis. The labile component, HIF-1α, is not only activated by hypoxia but also by peptides such as insulin and interleukin-1 (IL-1) in normoxia. We investigated whether inhibitors of mitogen-activated protein kinase kinases (MAPKKs: PD 98059, U0126) and phosphatidylinositol 3-kinase (PI3K: LY 294002) do not only lower the hypoxia-induced, but also the insulin- and IL-1-induced HIF-1α accumulation and HIF-1 DNA-binding in human hepatoma cell cultures (line HepG2). The results show that LY 294002 suppressed HIF-1 activation in a dose-dependent manner irrespective of the stimulus. With respect to target proteins controlled by HIF-1, the production of erythropoietin was fully blocked and that of vascular endothelial growth factor reduced following inhibition of the PI3K pathway. The role of MAPKKs in this process remained in question, because PD 98059 and U0126 did not significantly reduce HIF-1α levels at non-toxic doses. We propose that PI3K signaling is not only important in the hypoxic induction of HIF-1 but it is also crucially involved in the response to insulin and IL-1.