Recently it has been demonstrated that the single-copy malate synthase (MS) and isocitrate lyase (ICL) genes from cucumber are regulated by nutritional status in cucumber cell cultures. In this paper a new cucumber mesophyll protoplast transient expression system is described in which electroporated MS promoter-GUS reporter gene constructs exhibit the same pattern of expression as the endogenous MS gene. Both the electroporated MS-GUS constructs and the endogenous gene are expressed when protoplasts are cultured for 48 h on a non-metabolizable carbon source such as mannitol or 3-methylglucose, and repressed when cultured on a utilizable carbon source such as sucrose, glucose or fructose. A series of deletion mutants identified a region from position -248 to -125 relative to the start of transcription that is essential for expression of the MS-GUS construct under the different metabolic conditions. A 191 bp fragment spanning this region was fused, in both orientations, to the CaMV 35S core promoter. A pattern of metabolic regulation similar to that of the intact MS promoter was observed for these promoter fusion constructs which strongly suggests the presence of enhancer element(s) within this region. Comparison of the 191 bp region with other MS and ICL promoter sequences revealed a region of homology, designated RT. A gel retardation assay was used to assess binding of the 191 bp fragment to whole cell protein extracts from cell cultures expressing MS. Both the unlabelled 191 bp fragment and a synthetic oligonucleotide of RT compete specifically for the demonstrated binding activity.