The Human Ether-a-go-go Related Gene (hERG) encodes a delayed rectifier potassium channel that is widely expressed in neuronal, cancerous and cardiac tissue. In the heart, hERG channels contribute to the repolarisation of the action potential. Attenuation of hERG current, resulting from inherited mutations in the channel, or drug block, causes abnormal action potential repolarisation and cardiac arrhythmias, such as Torsades de Pointes. Arrhythmias are often precipitated by extreme emotion, stress or physical exertion, implying a role for the sympathetic nervous system. In this study we investigated hERG channel modulation and phosphorylation by protein kinase C (PKC). hERG current stably expressed in HEK cells was attenuated by 44 ± 3 % (n = 9) upon direct activation of PKC by 10 μM 1-oleoyl-2-acetylglycerol (OAG). The response to OAG could be inhibited by the PKC inhibitor bis-1, or by down-regulating PKC by chronic PMA treatment of cells. The hERG channel has 18 putative PKC phosphorylation sites. To investigate if PKC regulation occurs via direct phosphorylation of the channel, we measured phosphorylation of hERG by [³²P]-orthophosphate incorporation. Untreated hERG-HEK cells showed two bands at 155 and 135 kDa, corresponding to surface expressed, fully glycosylated, and core glycosylated forms of hERG respectively, indicating substantial levels of basal phosphorylation. OAG increased phosphorylation of both bands by 18 ± 2 % (p<0.001, n = 6). Surface protein experiments revealed equal amounts of hERG protein were immunoprecipitated in OAG-challenged and untreated cells. Currents from ΔPKC-hERG, a channel in which 17 of the 18 putative PKC phosphorylation sites have been mutated to Ala (Thomas et al., 2003) were also modulated with OAG, suggesting phosphorylation occurs at the 18^th site, T74 on the amino terminus, or at an atypical site. ΔPKC-hERG channels were not recognised by hERG antibodies and T74 mutants (T74A, V, D or E) were non-functional. Nevertheless, deletion of the N-terminus generated functional channels, but prevented modulation of channel activity and the increase in phosphorylation. Our results suggest PKC can modulate hERG currents through a mechanism that induces direct phosphorylation of the N-terminus. Further work will investigate if α₁ adrenergic receptor stimulation regulates hERG by a mechanism involving PKC-dependent phosphorylation.