The contributions of Na+/K+-ATPase, K+channels, and the NaK2Cl cotransporter (NKCC) to total and unidirectional K+flux were determined in mammalian skeletal muscle at rest. Rat hindlimbs were perfused in situ via the femoral artery with a bovine erythrocyte perfusion medium that contained either86Rb or42K, or both simultaneously, to determine differences in ability to trace unidirectional K+flux in the absence and presence of K+-flux inhibitors. In most experiments, the unidirectional flux of K+into skeletal muscle (JinK) measured using86Rb was 810% lower than JinK measured using42K. Ouabain (5 mM) was used to inhibit Na+/K+-ATPase activity, 0.06 mM bumetanide to inhibit NKCC activity, 1 mM tetracaine or 0.5 mM barium to block K+channels, and 0.05 mM glybenclamide (GLY) to block ATP-sensitive K+(KATP) channels. In controls, JinK remained unchanged at 0.31 ± 0.03 µmol·g1·min1during 55 min of perfusion. The ouabain-sensitive Na+/K+-ATPase contributed to 50 ± 2% of basal JinK, K+channels to 47 ± 2%, and the NKCC to 12 ± 1%. GLY had minimal effect on JinK, and both GLY and barium inhibited unidirectional efflux of K+(JoutK) from the cell through K+channels. Combined ouabain and tetracaine reduced JinK by 55 ± 2%, while the combination of ouabain, tetracaine, and bumetanide reduced JinK by 67 ± 2%, suggesting that other K+-flux pathways may be recruited because the combined drug effects on inhibiting JinK were not additive. The main conclusions are that the NKCC accounted for about 12% of JinK, and that KATPchannels accounted for nearly all of the JoutK, in resting skeletal muscle in situ.Key words: sodium potassium chloride cotransporter, NKCC, Na+/K+-ATPase, potassium channels, potassium transport, in situ rat hindlimb.