ATP-sensitive K⁺ (K_ATP) channels couple the metabolic state of a cell to its membrane potential. This property allows pancreatic β-cells to regulate insulin secretion in accordance with blood glucose levels. Genetic mutations associated with persistent hyperinsulinaemic hypoglycaemia of infancy (PHHI) and permanent neonatal diabetes mellitus (PNDM) have been shown to affect forward trafficking and endocytosis of pancreatic K_ATP channels respectively [1 & 2]. These findings indicate a possible role for changes in K_ATP channel cell surface density in the physiological and pathophysiological response of the β-cell to blood glucose fluctuations. Recent work from our laboratory has demonstrated that the pancreatic K_ATP channel undergoes rapid (t_0.5 ~ 5min) endocytosis in a constitutive fashion [2]. In addition, previous studies have found that inhibition of protein kinase C (PKC) leads to an increase in K_ATP channel cell surface density, an effect attributed to inhibition of channel endocytosis [3]. Here we report similar findings in the β-cell derived INS1e cell line. However, in addition to an inhibition of endocytosis it is possible that the increase in cell surface K_ATP channel density could be explained by increased channel recycling. As there were no data to support this hypothesis we first had to establish whether internalised K_ATP channels were able to recycle back into the plasma membrane. In order to investigate this possibility, K_ATP channels comprising SUR1 and extracellular HA-tagged Kir6.2 subunits were expressed in a variety of mammalian cell lines. Cell surface channels were allowed to bind and internalise anti-HA antibodies for 2 hrs at 37° C; after removing excess antibodies at 4° C, the cells were incubated at 37° C to permit recycling. Recycled and non-recycled channels were stained with fluorophore-conjugated secondary antibodies and visualised by confocal microscopy. Internalised K_ATP channels enter the perinuclear compartments enriched in the cation-independent mannose-6-phosphate receptor, from where they are rapidly recycled to the plasma membrane; this recycling is enhanced by the PKC inhibitor chelerythrine. Unexpectedly, however, endocytosis of K_ATP channels per se was unaffected by chelerythrine. Further studies using dominant negative constructs of Rab proteins demonstrated that chelerythrine inhibits exit of K_ATP channels from early endosomal compartments (EEA1 and Rab4 positive) into perinuclear compartments, promoting recycling of channels via the Rab4 dependent route. In conclusion, our data suggest that when PKC is activated, endocytosed K_ATP channels largely enter the perinuclear compartments and that their recycling back to the cell surface via a Rab4 dependent rapid recycling pathway is inhibited, thereby accounting for the observed decrease in cell surface channel density resulting from PKC activation.