The systemin precursor, prosystemin, has been previously shown to be sequestered in vascular bundles of tomato ( Lycopersicon esculentum Mill.) plants, but its subcellular compartmentalization and association with a specific cell type has not been established. We present in situ hybridization and immunocytochemical evidence at the light, confocal, and transmission electron microscopy levels that wound-induced and methyl jasmonate-induced prosystemin mRNA and protein are exclusively found in vascular phloem parenchyma cells of minor veins and midribs of leaves, and in the bicollateral phloem bundles of petioles and stems of tomato. Prosystemin protein was also found constitutively in parenchyma cells of various floral organs, including sepals, petals and anthers. At the subcellular level, prosystemin was found compartmentalized in the cytosol and the nucleus of vascular parenchyma cells. The cumulative data indicate that vascular phloem parenchyma cells are the sites for the synthesis and processing of prosystemin as a first line of defense signaling in response to herbivore and pathogen attacks.