Ethylene plays an important role in the regulation of many growth and developmental processes of higher plants. In tomato, Le-ACS6, a member of the ACC synthase multigene family involved in system 1 ethylene biosynthesis during fruit ripening, is subject to negative feedback regulation by ethylene. To identify the cis-elements that are responsible for the negative feedback control, we established an in vitro transient assay system employing particle bombardment on mature-green tomato fruit pericarp to examine the expression of a luciferase (LUC) reporter gene driven by a 5′-serially deleted Le-ACS6 promoter. The results localized putative cis-elements required for negative ethylene-response between −347 and −266 upstream from the translational start site ATG. Several lines of stable transformation of the Le-ACS6 promoter and GUS reporter fusion gene containing internal deletion from −347 to −266 were generated. The expression pattern of the GUS reporter showed that removal of the nucleotides from −347 to −266 completely eliminated the response of the Le-ACS6 promoter to exogenous ethylene.