α-Zingiberene synthase (ZIS), a sesquiterpene synthase gene that was isolated from lemon basil (Ocimum basilicum L.), encodes an enzyme that catalyzes the formation of α-zingiberene, and other sesquiterpenes, from farnesyl diphosphate. Transgenic tomato fruits overexpressing ZIS under the control of the fruit ripening-specific tomato polygalacturonase promoter (PG) accumulated high levels of α-zingiberene (224–1000 ng g−1 fresh weight) and other sesquiterpenes, such as α-bergamotene, 7-epi-sesquithujene, β-bisabolene and β-curcumene, whereas no sesquiterpenes were detected in non-transformed control fruits. The ZIS-transgenic fruits also produced monoterpenes, such as α-thujene, α-pinene, β-phellandrene and γ-terpinene (1–22 ng g−1 fresh weight), which were either not detected or were found only in minute concentrations in control fruits. Recombinant ZIS overexpressed in Escherichia coli catalyzed the formation of these monoterpenes from geranyl diphosphate. As the ZIS protein apparently lacks a transit peptide, and is localized in the cytosol, the production of monoterpenes in the transgenic tomatoes suggests that a pool of geranyl diphosphate is available in the cytosol. The phenotype of the ZIS-transgenic tomatoes was the same as that for wild-type tomatoes, with regard to plant vigor and shape, but transgenic plants exhibited a small decrease in lycopene content. This study thus showed that the synthesis of both mono- and sesquiterpenes can be enhanced by the ectopic expression of a single transgene in tomato fruit, and it further demonstrated the interconnection between the pools of terpenoid precursors in the plastids and the cytosol.