Recently, we reported that mutation A1529D in the domain (D) IV P-loop of the rat skeletal muscle Na(+) channel mu(1) (DIV-A1529D) enhanced entry to an inactivated state from which the channels recovered with an abnormally slow time constant on the order of approximately 100 s. Transition to this "ultra-slow" inactivated state (USI) was substantially reduced by binding to the outer pore of a mutant mu-conotoxin GIIIA. This indicated that USI reflected a structural rearrangement of the outer channel vestibule and that binding to the pore of a peptide could stabilize the pore structure (Hilber, K., Sandtner, W., Kudlacek, O., Glaaser, I. W., Weisz, E., Kyle, J. W., French, R. J., Fozzard, H. A., Dudley, S. C., and Todt, H. (2001) J. Biol. Chem. 276, 27831-27839). Here, we tested the hypothesis that occlusion of the inner vestibule of the Na(+) channel by the fast inactivation gate inhibits ultra-slow inactivation. Stabilization of the fast inactivated state (FI) by coexpression of the rat brain beta(1) subunit in Xenopus oocytes significantly prolonged the time course of entry to the USI. A reduction in USI was also observed when the FI was stabilized in the absence of the beta(1) subunit, suggesting a causal relation between the occurrence of the FI and inhibition of USI. This finding was further confirmed in experiments where the FI was destabilized by introducing the mutations I1303Q/F1304Q/M1305Q. In DIV-A1529D + I1303Q/F1304Q/M1305Q channels, occurrence of USI was enhanced at strongly depolarized potentials and could not be prevented by coexpression of the beta(1) subunit. These results strongly suggest that FI inhibits USI in DIV-A1529D channels. Binding to the inner pore of the fast inactivation gate may stabilize the channel structure and thereby prevent USI. Some of the data have been published previously in abstract form (Hilber, K., Sandtner, W., Kudlacek, O., Singer, E., and Todt, H. (2002) Soc. Neurosci. Abstr. 27, program number 46.12).