Synthesis of granulose was investigated in 15 solvent-producing Clostridium strains. Only one of the strains did not produce granulose. The structure of granulose in Clostridium acetobutylicum P262 consisted of a high-molecular-weight polyglucan containing only (1-->4) linked d-glucopyranose units. Biosynthesis of granulose in C. acetobutylicum P262 was dependent on ADPglucose pyrophosphorylase, and granulose synthase and mutants defective in granulose accumulation lacked either one or both enzyme activities. Granulose-positive revertants exhibited both enzyme activities. ADPglucose pyrophosphorylase and granulose synthase were not subject to allosteric control by metabolites. Granulose accumulation and the biosynthetic enzyme activities were initiated immediately before the pH breakpoint and were detected in cells only at the end of the exponential growth phase. Granulose accumulation did not occur under conditions of nitrogen limitation, excess carbon, or excess energy.