Hypoxia inducible factor 1α (HIF-1α), an essential transcriptional factor, is negatively regulated by two different types of oxygen and Fe2+-dependent HIF hydroxylases, proline hydroxylase (PHD) and factor inhibiting HIF (FIH), under normoxia. Iron chelators have therefore been used for inducing HIF-1α expression by inhibiting the hydroxylases. In this study, the iron chelators displayed differential effects for PHD and FIH in cells depending on their iron specificity and membrane permeability rather than their in vitro potencies. The membrane permeability of the strict Fe2+-chelator potentially inhibited both hydroxylases, whereas the membrane impermeable one showed no inhibitory effect in cells. In contrast, the depletion of the extracellular Fe3+ ion was mainly correlated to PHD inhibition, and the membrane permeable one elicited low efficacy for both enzymes in cells. The 3′-hydroxyl group of quercetin, a natural flavonoid, was critical for inhibition of intracellular hydroxylases. Since the 3′-methylation of quercetin is induced by catechol-O-methyl transferase, the enzyme may regulate the intracellular activity of quercetin. These data suggest that the multiple factors of iron-chelators may be responsible for regulating the intracellular activity HIF hydroxylases. J. Cell. Biochem. 114: 864–873, 2013. © 2012 Wiley Periodicals, Inc.