Background <p>Ethylene-responsive <i>cis</i>-regulatory elements are critical for ethylene-mediated gene regulation. Although spatial regulation of the gene expression response to ethylene in plants has been reported, the regulatory elements involved remain largely unidentified. Previously, we characterized an AC-rich element (ACE, 5′-CCTAAACCCCAAAACAATC-3′) from bean (<i>Phaseolus vulgaris</i>) that confers ethylene responsiveness in etiolated <i>Arabidopsis</i> seedlings. Its first 9-bp region (ACEcore, 5′-CCTAAACCC-3′) is sufficient to bind with nuclear proteins, prompting further investigation into its role in response to ethylene.</p> Methods and results <p>Through biochemical, molecular, and genetic analyses, we identified two functional ethylene-regulated elements derived from the ACEcore in transgenic <i>Arabidopsis</i>. ACEcore functions as a constitutive ethylene-responsive factor (ERF)-binding element (CEBE), interacting with ERF1 and ORA59 and driving constitutive β-glucuronidase (GUS) expression throughout seedlings. In contrast, a mutated version of CEBE (5′-ttcAAAttt-3′, mutated bases in lowercase) acts as an ethylene-responsive element with spatial bias (EREB). EREB conferred ACC (an ethylene precursor) responsiveness to both minimal 35&#xa0;S and native <i>ORA59</i> promoters. Importantly, EREB resulted in the ethylene-responsive expression of GUS with spatial bias in the seedlings. It mediated short-term (12&#xa0;h) ACC-induced GUS expression in primary leaves. However, long-term ACC exposure (9 days) increased (light) or induced (darkness) GUS expression in the roots but repressed it in the hypocotyls of the seedlings. This organ expression bias was not observed when ethylene perception was inhibited by AgNO₃.</p> Conclusions <p>CEBE, especially EREB, contributes to the spatial regulation of gene expression in the <i>Arabidopsis</i> response to ethylene, providing functional elements for ethylene-mediated transcriptional control.</p>