Selective K(+) transport in the inner mitochondrial membrane has been attributed to at least three different channel types: ATP-sensitive, Ca(2+)-regulated and voltage-dependent K(+) channels. Studies utilizing their selective modulators have suggested that an increased activity of these channels plays an important role in the cellular protection from metabolic stress. However, direct evidence for this effect is largely absent, and recent findings on the lack of specificity for several channel openers and blockers have questioned the actual contribution of the mitochondrial K(+) channels in the preservation of cellular viability. In order to directly investigate the role of enhanced mitochondrial K(+) uptake in cellular protection, we selectively expressed the inward rectifying K(+) channel Kir6.2 in the mitochondria of HEK293 and HL-1 cells. Targeted Kir6.2 expression was achieved by cloning the Kir6.2 gene in pCMV/mito/GFP vector and the proper trafficking to mitochondria was confirmed by colocalization studies and Western blot. An increased K(+) influx to mitochondria overexpressing Kir6.2, as evidenced by using the K(+)-sensitive PBFI AM fluorescent dye, substantially improved the cellular viability after hypoxic stress, which was assessed by lactate dehydrogenase (LDH) release. In parallel, monitoring of mitochondrial Ca(2+) during stress, via the specific indicator rhod-2, revealed a significant attenuation of Ca(2+) accumulation in mitochondria overexpressing K(+) channels. This effect was abolished in mitochondria expressing an inactive mutant of Kir6.2. Mitochondria expressing Kir6.2 K(+) channel also exhibited a significant degree of depolarization that became even more pronounced during the stress. In conclusion, this study provides the first non-pharmacological evidence that an increased K(+) influx to mitochondria protects against hypoxic stress by preventing detrimental effects of Ca(2+) overload.