While studying the adult rat skeletal muscle Na+ channel outer vestibule, we found that certain mutations of the lysine residue in the domain III P region at amino acid position 1237 of the alpha subunit, which is essential for the Na+ selectivity of the channel, produced substantial changes in the inactivation process. When skeletal muscle alpha subunits (micro1) with K1237 mutated to either serine (K1237S) or glutamic acid (K1237E) were expressed in Xenopus oocytes and depolarized for several minutes, the channels entered a state of inactivation from which recovery was very slow, i.e., the time constants of entry into and exit from this state were in the order of approximately 100 s. We refer to this process as "ultra-slow inactivation". By contrast, wild-type channels and channels with the charge-preserving mutation K1237R largely recovered within approximately 60 s, with only 20-30% of the current showing ultra-slow recovery. Coexpression of the rat brain beta1 subunit along with the K1237E alpha subunit tended to accelerate the faster components of recovery from inactivation, as has been reported previously of native channels, but had no effect on the mutation-induced ultra-slow inactivation. This implied that ultra-slow inactivation was a distinct process different from normal inactivation. Binding to the pore of a partially blocking peptide reduced the number of channels entering the ultra-slow inactivation state, possibly by interference with a structural rearrangement of the outer vestibule. Thus, ultra-slow inactivation, favored by charge-altering mutations at site 1237 in micro1 Na+ channels, may be analogous to C-type inactivation in Shaker K+ channels.