Slow inactivation (SI) determines the availability of voltage-gated sodium channels and contributes to the regulation of neuronal excitability and action potential firing patterns in the central nervous system. The neuronal sodium channel subtype Nav1.6 was suggested to play an important role in maintaining high firing frequencies, possibly due to a reduced SI compared to other sodium channel subtypes. With this study we characterized SI of WT Nav1.6 and introduced mutations that severely altered the channel's propensity to slow inactivate. Highly conserved residues in the S6 segment of DII and DIII were identified as SI-sensitive hot spots: V966C in DII and N1455A in DIII. WT and mutant channels were transiently expressed in ND7/23 cells and patch-clamped. Steady-state SI was assessed with prepulses of varying potentials lasting for one minute. The N1455A mutation greatly enhanced SI whereas the V966C mutation shifted its voltage-dependence in the depolarising direction by ~20 mV. The maximal reduction of channel availability was ~75% in all cases. Other gating properties, such as activation or fast inactivation, were affected to a much lesser extent. Our findings show that Nav1.6 does not completely slow-inactivate. SI can be severely altered by mutations in segment 6 of DII and III, thereby confirming the importance of these residues to SI. The results may help to develop a more detailed structural model of SI and the investigated mutants may provide a useful tool to elucidate the influence of Nav1.6 SI on firing patterns in sensory neurons.