Retinal pigment epithelial (RPE) cells express vascular endothelial growth factor (VEGF) in response to high glucose or hypoxia. We hypothesised that VEGF expression and secretion by RPE cells in high glucose and hypoxia are regulated by protein kinase C (PKC). Primary cultured RPE cells from Sprague–Dawley rats were growth-arrested for 48hr in 0.5% FBS in 5.6 or 30mm d-glucose. Cells were exposed to hypoxic conditions (<1% O 2 , 5% CO 2 ) for the last 15–18hr of growth-arrest. PKC -α, -β 1 , -δ, -ε, and -ζ were expressed by RPE cells and exposure to high glucose for 48hr had no effect on expression as demonstrated by Western immunoblotting. High glucose, hypoxia or VEGF stimulated translocation of a number of the PKC isozymes to the membrane or particulate fractions implying activation. In response to high glucose or acute phorbol myristate acetate (PMA) stimulation, VEGF mRNA analysed by RT-PCR was increased. Intracellular VEGF protein identified by immunoblotting and confocal immunofluorescence imaging was significantly increased by high glucose, hypoxia or acute PMA stimulation. Calphostin C or a specific inhibitor of PKC-ζ prevented high glucose-stimulated VEGF expression in high glucose. VEGF secretion, as measured by ELISA in the culture medium, was enhanced in hypoxia but not in high glucose. Following exposure of RPE cells to PMA for 24hr, PKC-δ was significantly down regulated, whereas PKC-α, -β, -ε and -ζ remained unchanged. Secretion of VEGF in normal or high glucose, or hypoxia was significantly reduced following treatment with PMA for 24hr but not with the PKC-ζ inhibitor. We conclude that in high glucose and hypoxia PKC isozymes are activated and are necessary for VEGF expression. Secretion of VEGF is enhanced in hypoxia and appears to be regulated by PKC-δ. RPE cells may contribute to the pathogenesis of retinopathy caused by high glucose and hypoxia through the expression and secretion of VEGF that are regulated by PKC isozymes.