We did not detect evidence of any known nicotine oxides with UPLC/ESI-QTOF-MS technology. We used third-instar larvae in contrast to the fifth-instar larvae used in the previous studies, which might differ physiologically from the first four growth-focused instars, as during the fifth instar, larvae prepare for pupation. Similar to the work of Self et al., we always fed the larvae immediately after eclosion on physiologically realistic concentrations of dietary nicotine, whereas Snyder et al. and Wink and Theile used the artificial diet-reared fifthinstar larvae, which had not been exposed to nicotine during their previous instars; these larvae were then abruptly exposed to high and physiologically unrealistic amounts of nicotine. We used freshly collected frass and hemolymph, whereas previous investigators used stored and dried frass in which nicotine oxidation may have occurred spontaneously. Lastly, we ruled out the possibility that nicotine was oxidized by the chemical constituents of diet, before it was ingested by the larvae, a possibility not examined in the previous work. None of the nicotine oxides were discharged from the hemolymph more rapidly than nicotine, clearly rejecting the long standing hypothesis that being more polar than nicotine, the oxides are more rapidly excreted. M. sexta larvae use pumps to purge toxic alkaloids to protect their nervous systems; these pumps function with structurally similar alkaloids like nicotine, morphine, atropine or even alkaline synthetic dyes. Previously, we proposed that MsCYP6B46 could be part of such a pump. The Waldbauer assays results were consistent with a ‘purge by excretion’ mechanism that functions with comparable efficiency between nicotine and its oxides. MsCYP6B46 transcripts were induced in larval midguts by the ingestion of all nicotine oxides, but no further oxidized products of nicotine, NNO or cotinine were found in the hemolymph or frass. Collectively, these results demonstrate either the absence of CYPmediated nicotine oxidation mechanisms in M. sexta, or that Manduca’s metabolism of nicotine is so rapid and efficient that no oxidative intermediates accumulate, or involves a novel pathway. All of the nicotine oxides had similar effects on larval growth as nicotine did and none deterred C. parallela, indicating that the oxidation of nicotine is not advantageous for M. sexta and hence cannot be considered either a detoxification or a co-option related process. The nicotine-elicited MsCYP6B46 clearly provides an ecological benefit for the larvae, due in large part to the volatility of nicotine. M. sexta larvae would clearly be at an ecological disadvantage if they converted nicotine to a less volatile and consequently, less spider-deterrent oxides. In contrast, for S. exigua, which is more sensitive to N. attenuata’s nicotine than M. sexta, oxidizing nicotine to cotinine, which is less lethal, represents true detoxification.