There are two enolase genes, ENO1 and ENO2, per haploid yeast genome. Expression of the ENO1 gene is quantitatively similar in cells grown on glucose or gluconeogenic carbon sources. In contrast, ENO2 expression is induced more than 20-fold in cells grown on glucose as the carbon source. cis-Acting regulatory sequences were mapped within the 5'-flanking region of the constitutively expressed yeast enolase gene ENO1. A complex positive regulatory region was located 445 base pairs (bp) upstream from the transcriptional initiation site which was required for ENO1 expression in cells grown on glycolytic or gluconeogenic carbon sources. A negative regulatory region was located 160 bp upstream from the transcriptional initiation site. Sequences required for the function of this negative regulatory element were mapped to a 38-bp region. Deletion of all or a portion of these latter sequences permitted glucose-dependent induction of ENO1 expression that was quantitatively similar to that of the glucose-inducible ENO2 gene. The negative regulatory element therefore prevents glucose-dependent induction of the ENO1 gene. Hybrid 5'-flanking regions were constructed which contained the upstream regulatory sequences of one enolase gene fused at a site upstream from the TATAAA box in the other enolase gene. Analysis of the expression of enolase genes containing these hybrid 5'-flanking region showed that the positive regulatory regions of ENO1 and ENO2 were functionally similar, as were the regions extending from the TATAAA boxes to the initiation codons. Based on these studies, we conclude that the negative regulatory element plays the critical role in maintaining the constitutive expression of the ENO1 structural gene in cells grown on glucose or gluconeogenic carbon sources.