BACKGROUND: N-malonyl 1-aminocyclopropane-1-carboxylic acid (MACC) is a major conjugate of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and may therefore play an important role in regulating ethylene production, as well as ethylene-independent ACC signalling. While the enzyme responsible for this derivatization, ACC malonyltransferase (AMT), has been studied in the past, its identity remains unknown. Methods to assay AMT activity are not well established, and no standardized assay has been described. RESULTS: We optimized an AMT activity assay and investigated the biological implications of AMT. This assay can be divided into three parts: total protein extraction, in vitro AMT activity assay, and MACC detection. For these three parts, different parameters were optimized and combined into an integrated and robust protocol. We used gas chromatography for the indirect detection of MACC, which was compared to a direct LC-MS approach, indicating that the GC-based method is a good alternative readily available to most labs studying ethylene. Next, we used this in vitro AMT activity assay to study the biological function of MACC formation. We observed an ontogenetic, tissue-specific and an ethylene-mediated feedback effect on AMT activity in tomato and Arabidopsis. The feedback of ethylene on AMT activity seems to be important to regulate ethylene production levels. CONCLUSIONS: The optimized and robust AMT activity assay presented here will enable other plant researchers to investigate the biochemistry of the ethylene biosynthesis pathway through ACC conjugation into MACC. Our AMT activity method was deployed both in tomato and Arabidopsis, and revealed that AMT activity is tightly controlled by ethylene itself in a tissue-specific way.