Abstract: Accumulation of glycogen and trehalose in nutrient-limited cultures of Saccharomyces cerevisiae is negatively correlated with specific growth rate. Additionally, conditions of glucose excess (i.e. growth limitation by nutrients other than glucose) are often implicated in high-level accumulation of these storage carbohydrates. The present study investigates how the identity of the growth-limiting nutrient affects accumulation of storage carbohydrates in cultures grown at a fixed specific growth rate. In anaerobic chemostat cultures (D = 0.10 h(-1)) of S. cerevisiae, the choice of the growth-limiting nutrient (glucose, ammonia, sulfate, phosphate or zinc) strongly affected storage carbohydrate accumulation. Glycogen contents of biomass from glucose- and ammonia-limited cultures were 10- to 14-fold higher than those of cells grown under the other three glucose-excess regimes. Trehalose levels were specifically increased under nitrogen-limited conditions. These results demonstrate that storage carbohydrate accumulation in nutrient-limited cultures of S. cerevisiae is not a generic response to glucose excess, but is instead strongly dependent on the identity of the growth-limiting nutrient. While transcriptome analysis of wild-type and msn2Deltamsn4Delta strains confirmed transcriptional upregulation of glycogen and trehalose biosynthesis genes mediated by Msn2p/Msn4p, transcriptional regulation could not quantitatively account for the drastic changes in storage carbohydrate accumulation. Activity assays of glycogen synthase and glycogen phosphorylase supported involvement of post-transcriptional regulation. Consistent with the high glycogen levels in ammonia-limited cultures, the ratio of glycogen synthase to glycogen phosphorylase was up to eight-fold higher than in the other glucose-excess cultures.