Tomato (Lycopersicum esculentum) is an important fruit crop in the Americas, southern Europe, the Middle East, and India, with increasing production in China, Japan, and Southeast Asia. It is amenable to producing pharmaceuticals, particularly for oral delivery; for many of the same reasons, it is a popular vegetable. Its fruit does not contain toxic substances and is palatable uncooked; it is easily processed; the plants are able to be propagated by seed or clonally by tip or shoot cuttings; the plants have a high yield of fruit; there is reasonable biomass and protein content; and they are easily grown under containment. This chapter describes Agrobacterium-mediated transformation of the tomato nucleus using cotyledons as explants. We have used this protocol to generate transgenic lines from several tomato cultivars expressing various genes of interest and selectable markers. We also provide protocols for molecular characterization of transgenic lines and batch processing tomato fruit.