Mechanisms that may regulate the storage of energy as triacylglycerol in Saccharomyces cerevisiae were examined. First, the kinetics of Dga1p, which mediates the majority of diacylglycerol esterification, the lone committed step in triacylglycerol synthesis, was measured in vitro. With an apparent K (m) of 17.0 mu M, Dga1p has higher affinity for oleoyl-CoA than the only S. cerevisiae acyltransferase previously kinetically characterized, Lpt1p. Lpt1p is a 1-acylglycerol-3-phosphate O-acyltransferase that produces phosphatidate, a precursor to diacylglycerol. Therefore, limiting triacylglycerol synthesis to situations of elevated acyl-CoA concentration is unlikely. However, Dga1p's apparent V (max) of 5.8 nmol/min/mg was 20 times lower than Lpt1p's. This supports Dga1p being rate limiting for TAG synthesis. Dga1p activity was not activated or inhibited when seven different molecules (e.g., ATP) which reflect cellular energy status were provided at physiological concentrations. Thus, allosteric regulation was not found. Coordination between triacylglycerol and glycogen synthesis was also tested. Yeast genetically deficient in triacylglycerol synthesis did not store more energy in glycogen and vice versa. Lastly, we tested whether genetically limiting energy storage in triacylglycerol, glycogen, steryl esters, or combinations of these will increase ethanol production efficiency. In nutrient-rich media containing 5 % glucose, solely limiting glycogen synthesis had the greatest affect, increasing ethanol production efficiency by 12 %. Since limiting glycogen synthesis only had a modest effect on growth in media containing 10 % ethanol, such genetic manipulation may improve commercial ethanol production.