Skeletal muscle K_ATP channel is crucial in preventing fiber damage and contractile dysfunctions, possibly by preventing large damaging ATP depletion. The objective of this study was to investigate changes in energy metabolism during fatigue in wild type and Kir6.2^-/- flexor digitorum brevis (FDB), the latter muscle lacking functional K_ATP channels. Fatigue was elicited with one tetanic contraction every sec. In contrast to wild type, Kir6.2^-/- FDB had significantly greater decreases in ATP and total adenylate levels during the last 2 min of the fatigue period. Glycogen depletion was greater in Kir6.2^-/- FDB for the first 60 sec but not by the end of the fatigue period while there was no difference in glucose uptake. The total amount of glucosyl units entering glycolysis was the same between wild type and Kir6.2^-/- FDB. During the first 60 sec, Kir6.2^-/- generated less lactate and more CO₂, while in the last 120 sec Kir6.2^-/- FDB stopped generating CO₂ and produced more lactate. The ATP generated during fatigue from phosphocreatine, glycolysis (lactate) and oxidative phosphorylation (CO₂) was 3.3-fold greater in Kir6.2^-/- than wild type FDB. Considering that ATP and total adenylate were significantly less in Kir6.2^-/- FDB, it is suggested that Kir6.2^-/- FDB had a greater energy deficit despite a greater ATP production, which was further supported by the fact that glucose uptake, lactate and CO₂ production were greater in Kir6.2^-/- FDB during the recovery period. It is thus concluded that a lack of functional K_ATP channels results in an impairment of energy metabolism.