Point mutations in the para-orthologous sodium channel α-subunit of the head louse (M815I, T917I, and L920F) are associated with permethrin resistance and DDT resistance. These mutations were inserted in all combinations using site-directed mutagenesis at the corresponding amino acid sequence positions (M827I, T929I, and L932F) of the house fly para-orthologous voltage-sensitive sodium channel α-subunit (Vssc1WT) gene and heterologously co-expressed with the sodium channel auxiliary subunit of house fly (Vsscβ) in Xenopus oocytes. The double mutant possessing M827I and T929I (Vssc1MITI/Vsscβ) caused a ∼4.0 mV hyperpolarizing shift and the triple mutant, Vssc1MITILF/Vsscβ, caused a ∼3.2 mV depolarizing shift in the voltage dependence of activation curves. Vssc1MITI/Vsscβ, Vssc1TILF/Vsscβ, and Vssc1MITILF/Vsscβ caused depolarizing shifts (∼6.6, ∼7.6, and ∼8.8 mV, respectively) in the voltage dependence of steady-state inactivation curves. The M827I and L932F mutations reduced permethrin sensitivity when expressed alone but the T929I mutation, either alone or in combination, virtually abolished permethrin sensitivity. Thus, the T929I mutation is the principal cause of permethrin resistance in head lice. Comparison of the expression rates of channels containing single, double and triple mutations with that of Vssc1WT/Vsscβ channels indicates that the M827I mutation may play a role in rescuing the decreased expression of channels containing T929I.