The Human ether a-go-go related gene (HERG) encodes a voltage gated potassium channel expressed in cardiac and neuronal tissues. Attenuation of cardiac HERG current, through either inhibition by drugs or inherited mutations leads to increased risk of arrhythmias. Second messengers such as cyclic AMP and several kinases modify HERG channel activity. We have previously shown that stimulating protein kinase C (PKC) by Gα_q/11 – coupled muscarinic receptor stimulation or elevation of intracellular [Ca²⁺] results in a sustained decrease of HERG current, a positive shift of activation and acceleration of deactivation kinetics (Cockerill et al., 2003). It has been suggested that PKC modulates HERG by an indirect mechanism, possibly involving phosphorylation of an auxillary subunit in the channel complex (Thomas et al., 2003). The aim of the present study was to determine if PKC activation results in increased phosphorylation of HERG stably expressed in HEK293 cells. Incorporation of ³²P into HERG was compared in non-treated cells and cells treated with 1-oleoyl-2-acetylglycerol (OAG) to activate PKC. HERG was immunoprecipitated, proteins were resolved by 10% SDS-PAGE and resulting blots subjected to autoradiography at -80^oC for 20h, as previously described (Budd et al., 1999). Phosphorylation of HERG was quantified using densitometry. Data are expressed as mean±S.E.M from ≥3 experiments. Statistical analysis was by unpaired Student’s t-tests with significance accepted at p<0.05.Two bands at 155 and 135kDa, corresponding to mature and core glycosylated forms of HERG respectively, were observed in untreated cells, indicatating phosphorylation under basal conditions. A 5min application of 10µM OAG significantly increased the phosphorylation of both bands by 18.1±1.7%. The OAG-dependent increase of phosphorylation could be abolished by preincubating cells with the PKC inhibitor bisindolylmaleimide-1 (3µM) for 15min or by down-regulating PKC by chronic treatment of cells with 1µM phorbol 12-myristate 13-acetate (PMA) for 24h. We have previously shown chronic PMA treatment to down-regulate the α,β,δ and ε isoforms of PKC, demonstrated using western blotting (Cockerill et al., 2003).Both methods of reducing PKC activity had no effect on HERG phosphorylation in non-OAG treated cells suggesting PKC is not responsible for basal phosphorylation of the channel. Our results suggest that HERG is highly phosphorylated under basal conditions and that stimulation of PKC with OAG increases the phosphorylation of the channel, suggesting that modulation of currents by PKC can occur by direct interactions with HERG.