methyltransferases (OMT) are important enzymes that are responsible for the synthesis of many small molecules, which include lignin monomers, flavonoids, alkaloids, and aroma compounds. One such compound is guaiacol, a small volatile molecule with a smoky aroma that contributes to tomato flavor. Little information is known about the pathway and regulation of synthesis of guaiacol. One possible route for synthesis is via catechol methylation. We identified a tomatoO-methyltransferase (CTOMT1) with homology to aNicotiana tabacumcatechol OMT.CTOMT1was cloned fromSolanum lycopersicumcv. M82 and expressed inEscherichia coli.Recombinant CTOMT1 enzyme preferentially methylated catechol, producing guaiacol. To validate thein vivofunction of CTOMT1, gene expression was either decreased or increased in transgenicS. lycopersicumplants. Knockdown ofCTOMT1resulted in significantly reduced fruit guaiacol emissions.CTOMT1overexpression resulted in slightly increased fruit guaiacol emission, which suggested that catechol availability might limit guaiacol production. To test this hypothesis, wild type (WT) andCTOMT1that overexpress tomato pericarp discs were supplied with exogenously applied catechol. Guaiacol production increased in both WT and transgenic fruit discs, although to a much greater extent inCTOMT1overexpressing discs. Finally, we identifiedS. pennelliiintrogression lines with increased guaiacol content and higher expression ofCTOMT1.These lines also showed a trend toward lower catechol levels. Taken together, we concluded that CTOMT1 is a catechol-O-methyltransferase that produces guaiacol in tomato fruit.