Volatile terpenoid compounds, potentially derived from carotenoids, are important components of flavor and aroma in many fruits, vegetables and ornamentals. Despite their importance, little is known about the enzymes that generate these volatiles. The tomato genome contains two closely related genes potentially encoding carotenoid cleavage dioxygenases, LeCCD1A and LeCCD1B. A quantitative reverse transcriptase-polymerase chain reaction analysis revealed that one of these two genes, LeCCD1B, is highly expressed in ripening fruit (4 days post-breaker), where it constitutes 0.11% of total RNA. Unlike the related neoxanthin cleavage dioxygenases, import assays using pea chloroplasts showed that the LeCCD1 proteins are not plastid-localized. The biochemical functions of the LeCCD1 proteins were determined by bacterial expression and in vitro assays, where it was shown that they symmetrically cleave multiple carotenoid substrates at the 9,10 (9′,10′) positions to produce a C14 dialdehyde and two C13 cyclohexones that vary depending on the substrate. The potential roles of the LeCCD1 genes in vivo were assessed in transgenic tomato plants constitutively expressing the LeCCD1B gene in reverse orientation. This over-expression of the antisense transcript led to 87–93% reductions in mRNA levels of both LeCCD1A and LeCCD1B in the leaves and fruits of selected lines. Transgenic plants exhibited no obvious morphological alterations. High-performance liquid chromatography analysis showed no significant modification in the carotenoid content of fruit tissue. However, volatile analysis showed a ≥50% decrease in β-ionone (a β-carotene-derived C13 cyclohexone) and a ≥60% decrease in geranylacetone (a C13 acyclic product likely derived from a lycopene precursor) in selected lines, implicating the LeCCD1 genes in the formation of these important flavor volatiles in vivo.