The blood-brain barrier (BBB) is a metabolic and physiological barrier important for maintaining brain homeostasis. The aim of these studies was to determine the role of protein kinase C (PKC) activation in BBB paracellular permeability changes induced by hypoxia and post-hypoxic reoxygenation using in vitro and in vivo BBB models. In rat brain microvessel endothelial cells (RMECs) exposed to hypoxia (1% O₂; 24 h), a significant increase in total PKC activity was observed, and this was reduced by post-hypoxic reoxygenation (95% room air/ 5% CO₂) for 2 h. The expression of PKCII, PKC, PKC, PKCµ and PKC also increased following hypoxia (1% O₂; 24 h) and these protein levels remained elevated following post-hypoxic reoxygenation (95% room air/ 5% CO₂; 2 h). Increases in the expression of PKC and PKC were also observed following post-hypoxic reoxygenation (95% room air/ 5% CO₂; 2 h). Moreover, inhibition of PKC with chelerythrine chloride (10 µM) attenuated the hypoxia-induced increases in ¹⁴C-sucrose permeability. Similar to what was observed in RMECs, total PKC activity was also stimulated in cerebral microvessels isolated from rats exposed to hypoxia (6% O₂; 1 h) and post-hypoxic reoxygenation (room air; 10 min). In contrast, hypoxia (6% O₂; 1 h) and post-hypoxic reoxygenation (room air; 10 min) significantly increased the expression levels of only PKC and PKC in the in vivo hypoxia model. These data demonstrate that hypoxia-induced BBB paracellular permeability changes occur via a PKC-dependent mechanism, possibly by differentially regulating the protein expression of the eleven PKC isozymes.