Cellulomonas flavigena strain KU (ATCC 53703) is a cellulolytic, Gram-positive bacterium which produces large quantities of an insoluble exopolysaccharide (EPS) when grown in minimal media with a high carbon-to-nitrogen (C/N) ratio. Earlier studies proved the EPS is structurally identical to the linear β-1,3-glucan known as curdlan and provided evidence that the EPS functions as a carbon and energy reserve compound. We now report that C. flavigena KU also accumulates two intracellular, glucose-storage carbohydrates under conditions of carbon and energy excess. These carbohydrates were partially purified and identified as the disaccharide trehalose and a glycogen/amylopectin-type polysaccharide. A novel method is described for the sequential fractionation and quantitative determination of all three carbohydrates from culture samples. This fractionation protocol was used to examine the effects of C/N ratio and osmolarity on the accumulation of cellular carbohydrates in batch culture. Increasing the C/N of the growth medium caused a significant accumulation of curdlan and glycogen but had a relatively minor effect on accumulation of trehalose. In contrast, trehalose levels increased in response to increasing osmolarity, while curdlan levels declined and glycogen levels were generally unaffected. During starvation for an exogenous source of carbon and energy, only curdlan and glycogen showed substantial degradation within the first 24 h. These results support the conclusion that extracellular curdlan and intracellular glycogen can both serve as short-term reserve compounds for C. flavigena KU and that trehalose appears to accumulate as a compatible solute in response to osmotic stress.