Trichoplusia ni caterpillars are polyphagous and rarely encounter aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus flavus and A. parasiticus in their host plants. To determine whether their ability to metabolize AFB1 varies with developmental stage, we evaluated the toxicity of AFB1 to T. ni caterpillars at different developmental stages and have demonstrated that all stages of T. ni larvae are much more tolerant to AFB1 than Helicoverpa zea, which as a fruit and seed feeder more frequently encounter AFB1 in its host plants. Bioactivation of AFB1 to its more toxic form is mediated by cytochrome P450 monooxygenases (P450s) as evidenced by the decrease of toxicity in the presence of piperonyl butoxide, a general P450 inhibitor. Some plant allelochemicals that have been shown to induce P450s ameliorate AFB1 toxicity suggesting that P450s are also involved in AFB1 detoxification in T. ni.