The Myc promoter-binding protein-1 (MBP-1) is a 37–38 kDa protein that binds to the c-myc P2 promoter and negatively regulates transcription of the protooncogene. MBP-1 cDNA shares 97% similarity with the cDNA encoding the glycolytic enzyme α-enolase and both genes have been mapped to the same region of human chromosome 1, suggesting the hypothesis that the two proteins might be encoded by the same gene. We show here data indicating that a 37 kDa protein is alternatively translated from the full-length α-enolase mRNA. This shorter form of α-enolase is able to bind the MBP-1 consensus sequence and to downregulate expression of a luciferase reporter gene under the control of the c-myc P2 promoter. Furthermore, using α-enolase/green fluorescent protein chimeras in transfection experiments we show that, while the 48 kDa α-enolase mainly has a cytoplasmic localization, the 37 kDa α-enolase is preferentially localized in the cell nuclei. The finding that a transcriptional repressor of the c-myc oncogene is an alternatively translated product of the ENO1 gene, which maps to a region of human chromosome 1 frequently deleted in human cancers, makes ENO1 a potential candidate for tumor suppressor.