This study investigated whether high frequency in vitro stimulation of rat fast-twitch extensor digitorum longus muscle depresses Na(+), K(+)-ATPase (NKA) activity as measured by the maximal in vitro 3-O-MFPase assay. EDL muscles subjected to 10 s continuous 100 Hz stimulation reduced tetanic force by 51.8 +/- 5.1% which recovered to 81.2 +/- 6.1% after 1 min and remained stable over 1 h recovery period. A second bout reduced force by 50.3 +/- 3.8% of initial but had no effect on 3-O-MFPase activity. Three minutes of intermittent stimulation (1 s at 100 Hz and 4 s recovery) resulted in 87.0 +/- 2.8% decline force with slow recovery (62.7 +/- 5.8% of initial after 1 h). The second 3-min bout reduced force by 83.3 +/- 3.6% of initial with no change in maximal 3-O-MFPase activity. These findings contrast previous human studies where fatiguing voluntary exercise depresses maximal NKA activity. This suggests that NKA in rat fast-twitch muscle is resistant to fatigue-induced inactivation under these conditions. Furthermore, the loss of force with fatigue was not related to depressed maximal NKA activity.