Oxygen-dependent regulation of HIF-1 activity occurs at multiple levels in vivo. The mechanisms regulating HIF-1 α 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-1 α a certainly play a role. Here, we show that ionomycin could activate HIF-1 transcriptional activity in a way that was additive to the effect of hypoxia without affecting HIF-1 α protein level. In addition, a calmodulin dominant negative mutant and W7, a calmodulin antagonist, 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. Furthermore, ERK but not JNK phosphorylation was evidenced in both conditions, ionomycin and hypoxia. PD98059, an inhibitor of the ERK pathway as well as a ERK1 dominant negative mutant also blocked HIF-1 activation by hypoxia and by ionomycin. A MEKK1 (a kinase upstream of JNK) dominant negative mutant had no effect. In addition, BAPTA, calmidazolium, a calmodulin antagonist and PD98059 inhibited VEGF secretion by hypoxic HepG2. All together, these results suggest that calcium and calmodulin would act upstream of ERK in the hypoxia signal transduction pathway. © 2002 Wiley-Liss, Inc.