Current evidence suggests that a few global regulatory factors mediate many of the extensive changes in gene expression that occur as Escherichia coli enters the stationary phase. One of the metabolic pathways that is transcriptionally activated in the stationary phase is the pathway for biosynthesis of glycogen. To identify factors that regulate glycogen biosynthesis in trans, a collection of transposon mutants was generated and screened for mutations which independently increase or decrease glycogen levels and the expression of a plasmid-encoded glgC'-lacZ fusion. The glycogen excess mutation TR1-5 was found to be pleiotropic. It led to increased expression of the genes glgC (ADPglucose pyrophosphorylase) and glgB (glycogen branching enzyme), which are representative of two glycogen synthesis operons, and the gluconeogenic gene pckA (phosphoenolpyruvate carboxykinase), and it exhibited effects on cell size and surface (adherence) properties. The mutated gene was designated csrA for carbon storage regulator. Its effect on glycogen biosynthesis was mediated independently of cyclic AMP (cAMP), the cAMP receptor protein, and guanosine 3'-bisphosphate 5'-bisphosphate (ppGpp), which are positive regulators of glgC expression. A plasmid clone of the native csrA gene strongly inhibited glycogen accumulation and affected the ability of cells to utilize certain carbon sources for growth. Nucleotide sequence analysis, complementation experiments, and in vitro expression studies indicated that csrA encodes a 61-amino-acid polypeptide that inhibits glycogen biosynthesis. Computer-assisted data base searches failed to identify genes or proteins that are homologous with csrA or its gene product.