HIF-1 (hypoxia-inducible factor-1) is the main transcription factor responsible for increased gene expression in hypoxia. The oxygen-dependent regulation of HIF-1 activity occurs at multiple levels in vivo. The mechanisms regulating HIF-1alpha protein expression have been most extensively analyzed, but the ones modulating HIF-1 transcriptional activity remain unclear. Changes in the phosphorylation and/or redox status of HIF-1alpha certainly play a role. Here, we show that ionomycin could activate HIF-1 transcriptional activity in a way that is additive to the effect of hypoxia without affecting HIF-1alpha protein level and HIF-1 DNA binding capacity. In addition, a calmodulin dominant-negative mutant as well as BAPTA, an intracellular calcium chelator, inhibited the hypoxia-induced HIF-1 activation. These results indicate that elevated calcium in hypoxia could participate in HIF-1 activation. PD98059, an inhibitor of the ERK pathway, but not KN-93, an inhibitor of calmodulin kinases II and IV, also blocked HIF-1 activation by hypoxia and by ionomycin. Altogether, these results suggest that calcium and calmodulin would act upstream of ERK in the hypoxia signal transduction pathway leading to enhanced HIF-1 transcriptional activity.