Tomato flavor is dependent upon a complex mixture of volatiles including multiple acetate esters. Red-fruited species of the tomato clade accumulate a relatively low content of acetate esters in comparison with the green-fruited species. We show that the difference in volatile ester content between the red-and greenfruited species is associated with insertion of a retrotransposon adjacent to the most enzymatically active member of a family of esterases. This insertion causes higher expression of the esterase, resulting in the reduced levels of multiple esters that are negatively correlated with human preferences for tomato. The insertion was evolutionarily fixed in the red-fruited species, suggesting that high expression of the esterase and consequent low ester content may provide an adaptive advantage in the ancestor of the redfruited species. These results illustrate at a molecular level how closely related species exhibit major differences in volatile production by altering a volatile-associated catabolic activity.T he flavor of a food involves integration of the information detected by taste and olfactory receptors (1). In the case of tomato (Solanum lycopersicum), flavor is the sum of the interactions between sugars, acids, and multiple volatile chemicals (2). Although sugars and acids are essential to our appreciation of tomato, the uniqueness and complexity of its flavor are dependent upon the blend of volatiles that can be detected (3, 4). Plants synthesize a vast array of volatile organic compounds. Derived from primary and secondary metabolites, they contribute to functions as diverse as defense against herbivores and pathogens, plant-toplant interactions, and attraction of pollinators and seed dispersers (5-7). Most of the important tomato volatiles are derived from amino acids, fatty acids, and carotenoid precursors (2,8). Despite the importance of these volatiles to fruit quality, regulation of their synthesis still remains poorly understood (4).One approach to identification of genes involved in plant volatile production is based upon characterization of quantitative trait loci (QTL). This method exploits variation between cultivars of the same species or between closely related species (9-11). In tomato, populations of introgression lines (ILs) that contain only a fragment of the genome of a wild species have been useful in the discovery of numerous volatile-associated QTL (10, 12) as well as of QTL affecting yield, carotenoid production, and accumulation of primary metabolites (13,14). From a human flavor perspective, it makes the most sense to concentrate on the volatiles that are most important to consumer preferences. Historically, the most important volatiles have been identified on the basis of odor units, that is, the concentration of a given volatile divided by its odor threshold (2, 15). Although this method has value, recent work has shown that the reality of taste preference is far more complex as interactions between volatiles and other flavor-associated chemicals influence perception....