When muscle fibers are repeatedly stimulated, they may become depolarized and force output decline. Excitation of the transverse tubular system (T-system) is critical for activation, but its role in muscle fatigue is poorly understood. Here, mechanically skinned fibers from rat fast-twitch muscle were used, because the sarcolemma is absent but the T-system retains normal excitability and its properties can be studied in isolation. The T-system membrane was fully polarized by bathing the skinned fiber in an internal solution with 126 mM K(+) (control solution) or set at partially depolarized levels (approximately -63 and -58 mV) in solutions with 66 or 55 mM K(+), respectively, and action potentials (APs) were triggered in the sealed T-system by field stimulation. Prolonged depolarization of the T-system reduced tetanic force proportionately more than twitch force, with greater effect at higher stimulation frequency (responses at 20 and 100 Hz reduced to 71 and 62% in 66 mM K(+) and to 54 and 35% in 55 mM K(+), respectively). Double-pulse stimulation showed that depolarization increased the repriming period (estimated minimum time before a second AP can be produced) from approximately 4 ms to approximately 7.5 and 15 ms in the 66 and 55 mM K(+) solutions, respectively. These results demonstrate that T-system depolarization reduces tetanic force by impairing AP repriming, rather than by preventing AP generation per se or by inactivating the T-system voltage sensors. The findings also explain why it is advantageous to reduce the rate of motoneuron stimulation to muscles during repeated or prolonged periods of activity.