The spatial distribution of plant defenses within a leaf may be critical in explaining patterns of herbivory. The generalist lepidopteran larvae, Helicoverpa armigera (the cotton bollworm), avoided the midvein and periphery of Arabidopsis thaliana rosette leaves and fed almost exclusively on the inner lamina. This feeding pattern was attributed to glucosinolates because it was not evident in a myrosinase mutant that lacks the ability to activate glucosinolate defenses by hydrolysis. To measure the spatial distribution of glucosinolates in A. thaliana leaves at a fine scale, we constructed ion intensity maps from MALDI-TOF (matrix assisted laser desorption/ionization-time of flight) mass spectra. The major glucosinolates were found to be more abundant in tissues of the midvein and the periphery of the leaf than the inner lamina, patterns that were validated by HPLC analyses of dissected leaves. In addition, there were differences in the proportions of the three major glucosinolates in different leaf regions. Hence, the distribution of glucosinolates within the leaf appears to control the feeding preference of H. armigera larvae. The preferential allocation of glucosinolates to the periphery may play a key role in the defense of leaves by creating a barrier to the feeding of chewing herbivores that frequently approach leaves from the edge.antiherbivore defense ͉ MALDI-imaging ͉ plant natural products M any plant natural products appear to serve as defenses against herbivores because of their toxicity or deterrence in artificial diets or when added as a supplement to plant material (1, 2). However, to evaluate the actual defensive role of these substances in planta, it is necessary to estimate the amount that a potential herbivore would encounter during feeding. Although determining the amount of plant tissue ingested during a feeding bout is relatively straightforward, measuring the quantity of specific defense products in that tissue is not. Nearly all studies to date have quantified the levels of defensive metabolites at the level of the whole plant or organ (3). Little is known about the localization of antiherbivore defenses in individual tissues or parts of organs even though this may be critically important to the behavior of small herbivores and to the effectiveness of the defense.One of the most extensively studied classes of antiherbivore chemical defenses in plants is the glucosinolates, a group of sulfur-rich, amino acid-derived metabolites combining a -Dglucopyranose residue linked via a sulfur atom to an Nhydroxyamino sulfate ester (4) (Fig. 1). Glucosinolates are widespread in the order Capparales, which includes vegetables (cabbage, cauliflower, and broccoli), spice plants supplying condiments (mustard, horseradish, and wasabi), and the model species, Arabidopsis thaliana (5, 6). Upon insect feeding or mechanical disruption, glucosinolates are hydrolyzed by an endogenous glucohydrolase activity known as myrosinase, and the released aglycone rearranges to form isothiocyanates, nitriles, and other pr...