Neuronal degeneration and aberrant neovascularization are common problems of ischemic retinopathies. Pigment epithelium-derived factor (PEDF), a neuroprotective protein and an inhibitor of angiogenesis, is produced by retinal glial (Müller) cells and can counterbalance elevated levels of vascular endothelial growth factor (VEGF), the expression of which is regulated primarily by hypoxia-inducible factor (HIF)-1. In an approach to mimic transient ischemia in vitro, primary Müller cells were cultured under transient and strong hypoxia (0.2% O2), followed by reoxygenation at 2.5% O2, and molecular mechanisms that might contribute to changes in the intraretinal PEDF level were determined. Hypoxic conditions caused an increasing expression of HIF-1α and led to upregulation of both PEDF and VEGF. Treatment of the cells with synthetic HIF-1α blockers or neutralization of VEGF binding to VEGF receptors (VEGFR-1 and-2) suppressed hypoxia-induced PEDF upregulation. Furthermore, the presence of CoCl2 (a hypoxia mimetic) induced an accumulation of elevated HIF-1α protein in the nucleus and an upregulation of PEDF expression in Müller cells. Increasing PEDF expression was attenuated when HIF-1α levels were suppressed using HIF-1α small interfering RNA (siRNA). On the other hand, siRNA-mediated depletion of PEDF facilitated HIF-1α upregulation caused by CoCl2 and resulted in increasing VEGF mRNA and protein levels. These results demonstrate that VEGF and PEDF may be unidirectionally regulated in hypoxia through HIF-1α activation, with upregulation of PEDF, which may occur in a VEGF-dependent manner. However, endogenously produced PEDF seems to be an inherent control element of HIF-1α expression in Müller cells, indicating an important feedback mechanism for limiting upregulation of VEGF. © 2011 Wiley Periodicals, Inc.