The iron-chelator desferrioxamine (DFO) and the transition metal cobalt induce hypoxia-inducible factor-1α (HIF-1α) in normoxia. DFO stabilizes HIF-1α from proteolysis by inhibiting the activity of iron-dependent prolyl hydroxylases, but the mechanism of action of cobalt is not fully elucidated. The purpose of this study was to examine the regulation of HIF-1α induction and HeLa cell proliferation by cobalt and the role of iron in these processes. Our results show that, unlike DFO, induction of transcriptionally active HIF-1α by CoCl₂ cannot be abrogated by the addition of excess Fe³⁺, but involves the production of reactive oxygen species (ROS) and the operation of the phosphatidylinositol-3 kinase (PI-3K) and MAPK pathways. CoCl₂, as well as DFO, decreased HeLa cell proliferation, but these effects were reversed by the addition of Fe³⁺. We conclude that the effect of cobalt on cell proliferation is iron-dependent, while its effects on HIF-1α induction are ROS- and signaling pathways-dependent, but iron-independent.