The aim of the present study was to examine fibre type-specific Na(+)-K(+) pump subunit expression and exercise-induced alterations in phospholemman (FXYD1) phosphorylation in humans. Segments of human skeletal muscle fibres were dissected and fibre typed, and protein expression was determined by Western blotting. The protein expression of the Na(+)-K(+) pump α2 isoform was lower in type I than in type II fibres (0.63 ± 0.04 a.u. vs. 1.00 ± 0.07 a.u., P < 0.001), while protein expression of the Na(+)-K(+) pump α1 and β1 isoforms was not different. Protein expression of the ATP-dependent potassium channel Kir6.2 was higher in type I compared with type II fibres. In both type I (P < 0.01) and type II fibres (P < 0.001) the AB_FXYD1 signal was lower after exercise compared with rest, indicating an increase in unspecific FXYD1 phosphorylation. The FXYD1 serine 68 phosphorylation was higher (P < 0.001) after exercise compared with rest in type II fibres (1.90 ± 0.25 vs. 1.00 ± 0.08) and not changed in type I fibres. Total FXYD1 was not expressed in a fibre type-specific manner. Expression of phosphofructokinase was lower (P < 0.001) in type I than in type II fibres, whereas citrate synthase and 3-hydroxyacyl-CoA dehydrogenase were more abundant (P < 0.001) in type I fibres. In conclusion, FXYD1 phosphorylation at serine 68 increased after an acute bout of intense exercise in human type II fibres, while AB_FXYD1 signal intensity was lower in both type I and type II fibres, indicating fibre type-specific differences in FXYD1 phosphorylation on serine 63, serine 68 and threonine 69. This, together with the observation of a higher abundance of the Na(+)-K(+) pump α2 isoform protein in type II fibres, is likely to have importance for the exercise-induced human Na(+)-K(+) pump activity in the different fibre types.