The K_ATP channel is an octameric protein complex composed of a pore-forming subunit K_IR (Kir6.0) and regulatory subunit, the sulphonylurea receptor (SUR). Kir6.1 is proposed to be part of the mitoK_ATP channel. The distribution of the Kir6.1 was investigated using fluorescent markers in immortalised cell lines. The C terminus of Kir6.1 was tagged to green fluorescent protein (GFP) using standard molecular biology techniques. HEK-293 and C2C12 cells were transiently transfected with Kir6.1-GFP and an endoplasmic reticulum (ER) marker called ErRed using the liposome based method. Cells were also cotransfected with SUR1. The distribution of Kir6.1 was visualised 36-48 h after transfection using confocal microscopy. The proportion of colocalisation was measured using laserpix software. This calculated the total number of existing green pixels (6.1GFP) that also contained red pixels (Er Red). The results were given as percentages (mean ± S.E.M.) and the Student’s t test was used to compare the proportion of colocalisation against a theoretical value of zero. The values obtained were from at least 6 separate transfections. Kir6.1 was found to be predominately located in the ER in both cell lines. C2C12 cells co-transfected with Kir6.1GFP and ErRed showed that about 36 ± 0.025% of Kir6.1 was retained in the ER (n=48) with 36 ± 0.028% also observed in cells co-transfected with SUR1 (n=32). In HEK-293 cells, more Kir6.1 was retained in the ER at 50 ± 0.029% (n=34). Again, there was no change in the distribution of Kir6.1 at 49 ± 0.012% when cells were cotransfected with SUR1 (n=48). Immunofluorescence studies detected the presence of endogenous Kir6.1 in these cell lines using antibodies specific to Kir6.1 and ER marker, concanavalin A. Controls were set up using a preimmune sample and antibody bound onto a fusion protein to which it was raised. In C2C12s, 36 ± 0.022% of Kir6.1 was found in the ER (n=25), with 49 ± 0.020% in HepG2s (n=30) and 45 ± 0.021% in HL-1 cardiomyocytes (n=28). No Kir6.1 was detected in HEK-293 cells. Colocalisation studies were carried out to see if Kir6.1 existed in the mitochondria. Cells were transfected before they were loaded with 150nm Mitotracker Red. However, the proportion of colocalisation is significantly lower in this experiment in comparison to colocalisation experiment with ErRed. About 26 ± 0.016% of Kir6.1 was found in mitochondria (n=36) and the percentage is not significantly different in the presence of SUR1 28 ± 0.019% (n=17). We conclude that the ATP-sensitive potassium subunit Kir6.1 is predominately distributed in the ER in cardiac and skeletal muscle cell lines, Kir6.1 is also retained in the ER when co-expressed the sulphonylurea receptor. These data show that a small percentage of Kir6.1 may exist in other locations such as the mitochondria.