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.