The inducibility of cytochrome P450 monooxygenases (P450s) and other xenobiotic-metabolizing enzymes is thought to reflect material and energy costs of biosynthesis. Efforts to detect such costs of detoxification enzyme induction, however, have had mixed success. Although they are rarely considered, ecological costs of induction may be a more significant evolutionary constraint on herbivores than material and energy costs. Because some P450-mediated metabolic transformations are bioactivation reactions that increase, rather than reduce, toxicity, maintaining high levels of P450 activity places an organism at risk of greater mortality in the presence of compounds that are bioactivated. We show that P450 inducibility in the generalist moth Helicoverpa zea in response to plant signaling substances, an adaptive response in a ditrophic interaction between herbivore and plant, becomes detrimental in the presence of a third trophic association with a plant pathogen that produces aflatoxin, a toxin that can be bioactivated by P450s. Consumption of plant signaling molecules, such as methyl jasmonate (MeJA) and salicylic acid (SA) enhanced the toxicity of aflatoxin B1 (AFB1) to H. zea that resulted in substantially more damage to larval growth and development. Among the P450 transcripts already cloned from this organism, two in the CYP6B and CYP321A subfamilies are shown to be induced in response to MeJA and SA, suggesting that they may mediate some of the observed bioactivations.