The NAD-dependent malic enzyme from Schizosaccharomyces pombe catalyzes the oxidative decarboxylation of L-malate to pyruvate and CO 2 . Transcription of the S. pombe malic enzyme gene, mae2, was studied to elucidate the regulatory mechanisms involved in the expression of the gene. No evidence for substrate-induced expression of mae2 was observed in the presence of 0.2% L-malate. However, transcription of mae2 was induced when cells were grown in high concentrations of glucose or under anaerobic conditions. The increased levels of malic enzyme may provide additional pyruvate or assist in maintaining the redox potential under fermentative conditions. Deletion and mutation analyses of the 5-flanking region of the mae2 gene revealed the presence of three novel negative cis-acting elements, URS1, URS2, and URS3, that seem to function cooperatively to repress transcription of the mae2 gene. URS1 and URS2 are also present in the promoter region of the S. pombe malate transporter gene, suggesting co-regulation of their expression. Furthermore, two positive cisacting elements in the mae2 promoter, UAS1 and UAS2, show homology with the DNA recognition sites of the cAMP-dependent transcription factors ADR1, AP-2, and ATF (activating transcription factor)/CREB (cAMP response element binding).The fission yeast Schizosaccharomyces pombe efficiently degrades L-malate to CO 2 under aerobic conditions and to ethanol and CO 2 under anaerobic conditions (1). Cells of S. pombe are not able to utilize malate as the sole energy source or incorporate the malate into biomass (2) and therefore require glucose or other carbon sources for the energy-dependent transport and efficient degradation of malic acid (3). Three enzymes are involved in malate degradation in S. pombe, namely the malate transporter, malic enzyme, and malate dehydrogenase (4). The transporter, encoded by the mae1 gene (5), uses an H ϩ -symport system for the active transport of L-malate, and the NAD-dependent malic enzyme (EC 1.1.1.38) catalyzes the oxidative decarboxylation of L-malate to pyruvate and CO 2 . The mitochondrial malate dehydrogenase oxidizes L-malate to oxaloacetate in the tricarboxylic acid cycle and is responsible for 10% of the degradation of malate under aerobic conditions. Molecular analysis of the S. pombe malic mae2 gene showed a high degree of homology with malic enzymes from various organisms (6). Eight highly conserved regions were identified in malic enzymes, including the binding sites for L-malate and the dinucleotide co-factors NAD(P) ϩ (7,8). Although the secondary structure of malic enzymes is highly conserved, the coenzyme specificity (NAD ϩ or NADP ϩ ) and cellular localization (cytosolic or mitochondrial) are strongly linked to their regulation and metabolic function (9). Cytosolic NADP-dependent malic enzymes play an important role in lipid metabolism in higher eukaryotes (10), whereas NAD-dependent malic enzymes provide mitochondrial NADH for electron transport or cytosolic NADH for reductive power for other metabolic pathways (9)....