Hyperglycaemia causes a marked vasoconstriction in resistance arteries. Protein Kinase C (PKC) activation has been demonstrated to be activated by an increase in extracellular glucose in a number of cell types including cardiac myocytes, renal tissue and vascular smooth muscle. We have previously identified that elevated extracellular glucose caused a marked inhibition of Kv currents in mesenteric arterial smooth muscle cells. Here we present data showing that increasing extracellular glucose concentration causes a marked concentration-dependent inhibition of Kv currents that can be reversed using selective PKCα and β inhibitors. Rat mesenteric arterial smooth muscle was enzymatically isolated as previously described (Rainbow et al 2006). All animals were humanely culled in accordance with Home Office guidelines. Using patch clamp electrophysiology, a concentration response curve of the effects of extracellular glucose on Kv current density was constructed. A current voltage (IV) relationship was recorded from -40 to +60 mV using 400 ms pulses in 10 mV steps from a holding potential of -70 mV. This was recorded initially in a solution containing 0 mM glucose and was followed by 10 minutes of perfusion with either 2, 5, 8, 10, 12, 15, 18, 20 or 30 mM glucose (all osmotically balanced to 30 mM with mannitol) when a second IV curve was recorded and the mean current over the final 100 ms of the pulse to +60 mV was measured and compared to that in 0 mM glucose. At least 6 recordings were made for each concentration across a minimum of 4 animals. Data showed a marked inhibition of current best fitted with a biphasic curve giving a top half IC50 value of 5.2 mM and a lower half of 14.2 mM. Using Western blotting we identified that PKCα, β, γ, δ and ε were present in mesenteric smooth muscle cell lysates (n=5 blots from 5 animals). In whole cell recording, inhibiting PKCα and β with either Go6976 or cell permeant Tat-peptide linked isoform-specific inhibitor peptides (Tat-PKC) attenuated the inhibition of Kv current by 20 mM glucose (fractional current 0.52±0.04 in control, 0.95±0.07*** in Go6976, 0.78±0.06* in Tat-PKCα, 0.85±0.09** in Tat-PKCβ and 0.94±0.03*** with both Tat-PKCα and Tat-PKCβ inhibitor present, all n=6, *P<0.05, **P<0.01, ***P<0.001, (ANOVA, Bonferroni)). Selective inhibition of PKCγ, δ or ε did not significantly affect the glucose-induced Kv inhibition (0.51±0.7, 0.59±0.8 and 0.61±0.9, all n=4, P>0.05 (ANOVA, Bonferroni)). These data suggest that PKCα and β are activated by elevated extracellular glucose and can act to inhibit Kv channels contributing to the glucose-induced depolarisation. Despite PKCε having been shown to inhibit Kv current in other studies (Rainbow et al 2009), inhibition of PKCγ, δ or ε had no effect on the inhibition of Kv by elevated extracellular glucose.