The human ether-a-go-go related gene (hERG) potassium channel is expressed in a variety of tissues including the heart, neurons and some cancer cells. hERG channels are modulated by several intracellular signalling pathways and these provide important mechanisms for regulating cellular excitability. In this study, we investigated muscarinic modulation of hERG currents and direct phosphorylation of channel subunits expressed in HEK-293 cells at physiologically relevant temperatures, by protein kinase C (PKC). Activation of G_q/11-coupled M₃-muscarinic receptors with methacholine, reduced current amplitudes at all potentials with minor effects on the voltage dependence of activation and inactivation. The response to methacholine was insensitive to intracellular BAPTA, but was attenuated by either acute inhibition of PKC with 300 nM bisindolylmaleimide-1 (bis-1) or chronic down-regulation of PKC isoforms by 24 hour pre-treatment of cells with phorbol 12-myristate 13-acetate (PMA). Stimulation of PKC with 1-oleoyl 2-acetyl glycerol (OAG), an analogue of diacylglycerol (DAG), mimicked the actions of muscarinic receptor stimulation. Direct phosphorylation of hERG was measured by [³²P]-orthophosphate labelling of immunoprecipitated protein with an anti-hERG antibody. Basal phosphorylation was high in unstimulated cells and further increased by OAG. The OAG dependent increase was abolished by bis-1 and down-regulation of PKC, but basal levels of phosphorylation were unchanged. Deletion of the amino-terminus of hERG prevented both the modulation of channel activity and the increase of phosphorylation by OAG. Our results are consistent with calcium and/or DAG sensitive isotypes of PKC modulating hERG currents through a mechanism that involves direct phosphorylation of sites on the amino terminus of hERG.