A southern house mosquito Culex quinquefasciatus Say colony was established from surviving late instars and pupae from a semifield evaluation on Natular XRG (a granular formulation containing 2.5% spinosad). The initial lethal levels of Natular XRG against this colony were determined in the laboratory for the first-generation progeny (designated as F₁). Selection pressure was applied at LC_70–90 levels to 10,000–15,000 late third- and early fourth-instar larvae of each generation with Natular XRG. Susceptibility changes in response to selection were determined every other generation, where a gradual and steady decline in susceptibility occurred from generation F₁ to F₃₅, followed by significant decline from generations F₃₇ to F₄₅ For reference purposes, susceptibility of freshly collected wild populations as well as a laboratory colony of the same species was also determined concurrently, which fluctuated within a slightly wider range for the wild populations and a tighter range for the laboratory colony. By comparing with wild populations and laboratory reference colony, tolerance to spinosad was observed from generations up to F₉ in the selected population. Resistance levels increased gradually from generation F₁₁ to F₃₅, and elevated significantly from generations F₃₇ to F₄₅ when resistance ratios reached 1,415.3- to 2,229.9-fold at LC₅₀ and 9,613.1- to 17,062.6-fold at LC₉₀ Possible mechanisms of resistance development to spinosad were discussed.