TASK-3 (KCNK9 or K2P9.1) channels are thought to promote proliferation and/or survival of malignantly transformed cells, most likely by increasing their hypoxia tolerance. Based on our previous results that suggested mitochondrial expression of TASK-3 channels the objective of the present work was to seek the effect of reduced TASK-3 biosynthesis on the mitochondrial function and survival of cultured melanoma cells.To achieve this, a stably transfected TASK-3 knockdown of primary tumor originated WM35 melanoma cell line was produced. Non-transfected and scrambled shRNA-transfected melanoma cells served as control. The effectiveness of gene silencing was monitored by RT-PCR and Western blot while the changes in different cell processes were analysed using MTT and CyQuant colorimetric assays and immunolabelling. Mitochondrial membrane potential (MMP) was monitored using the fluorescent dies JC-1 and DiOC6, while the reactive oxygen species (ROS) production was investigated with a superoxide indicator dihydroethidium (HE) dye. Values are given as means +/- S.E.M.TASK-3 knockdown cells presented reduced viability, decreased DNA content, altered cell morphology, and reduced surface area. In contrast to the non- and scrambled-shRNA-transfected melanoma cell lines, which did not present noteworthy apoptotic activity, almost 50% of the TASK-3 knockdown cells exhibited strong annexin V-specific immunofluorescence signal. In addition, apoptosis inducing factor (AIF), which normally localises in the mitochondrial matrix and released during apoptosis showed translocation to the nuclear region in TASK-3 gene silenced melanoma cells. Furthermore, TASK-3 knockdown WM35 cells had depolarized MMP, and they demonstrated diminished responsiveness to the application of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) compared to their control counterparts as measured using JC-1 dye. In TASK-3 knockdown cells we detected significantly decreased DiOC6 (39.9 +/- 0.8, a.u., n=9) and HE (107.2 +/- 4.8, a.u., n=9) fluorescence intensity with a FACS-Calibur flow cytometer compared with non-transfected and scrambled controls (75.9 +/- 1.5 and 80.9 +/- 1.8 a.u., n=9 in DiOC6 loaded cells) and (139.8 +/- 3.8 and 147.5 +/- 3.7, a.u., n=9 following HE incubation) respectively, indicating reduced mitochondrial membrane potential and ROS production. Interference with TASK-3 channel expression, therefore, induces apoptosis of cultured melanoma cells, most likely via causing mitochondrial depolarization.We conclude that TASK-3 channels are essential in maintaining mitochondrial function in WM35 melanoma cells. Our present findings suggest that TASK-3 channels may be legitimate targets of future melanoma-specific therapies.