A 1018 bp fragment of the ACO gene cDNA sequence was cloned from tomato (Lycopersicon esculentum) leaves incubated with a pathogen mixture using reverse transcriptase-polymerase chain reaction (RT-PCR) with two PCR primers designed according to the sequence of a tomato cDNA clone (E11). A BLAST search showed the sequence presenting a very high match with the ACO genes in other plants, with 83–99% homology. Using this sequence, an RNA interference (RNAi) transformation vector (pD311) was constructed and transformed into tomato. Twenty-seven regenerated plants with kanamycin resistance were obtained, showing that the transgene was integrated into the tomato genome; this was confirmed by PCR and Southern blotting. Ethylene production by the RNAi transgenic tomato plants was measured by gas chromatography, and showed that ethylene evolution was specifically inhibited in leaves and fruits of the transgenic plants.