Hypoxia-inducible factor-1 (HIF-1) controls the expression of a number of genes such as vascular endothelial growth factor and erythropoietin in low oxygen conditions. However, the molecular mechanisms that underlie the activation of the limiting subunit, HIF-1alpha, are still poorly resolved. Results showing that endogenous HIF-1alpha migrated 12 kDa higher than in vitro translated protein led us to evaluate the possible role of phosphorylation on this phenomenon. We report here that HIF-1alpha is strongly phosphorylated in vivo and that phosphorylation is responsible for the marked differences in the migration pattern of HIF-1alpha. In vitro, HIF-1alpha is phosphorylated by p42 and p44 mitogen-activated protein kinases (MAPKs) and not by p38 MAPK or c-Jun N-terminal kinase. Interestingly, p42/p44 MAPK stoichiometrically phosphorylate HIF-1alpha in vitro, as judged by a complete upper shift of HIF-1alpha. More importantly, we demonstrate that activation of the p42/p44 MAPK pathway in quiescent cells induced the phosphorylation and shift of HIF-1alpha, which was abrogated in presence of the MEK inhibitor, PD 98059. Finally, we found that in a vascular endothelial growth factor promoter mutated at sites previously shown to be MAPK-sensitive (SP1/AP2-88-66 site), p42/p44 MAPK activation is sufficient to promote the transcriptional activity of HIF-1. This interaction between HIF-1alpha and p42/p44 MAPK suggests a cooperation between hypoxic and growth factor signals that ultimately leads to the increase in HIF-1-mediated gene expression.