The RNA-binding protein CsrA (carbon storage regulator) is a new kind of global regulator, which facilitates specific mRNA decay. A recombinant CsrA protein containing a metal-binding affinity tag (CsrA-H6) was purified to homogeneity and authenticated by N-terminal sequencing, matrix-assisted laser desorption/ionization time of flight mass spectrometry, and other studies. This protein was entirely contained within a globular complex of approximately 18 CsrA-H6 subunits and a single approximately 350-nucleotide RNA, CsrB. cDNA cloning and nucleotide sequencing revealed that the csrB gene is located downstream from syd in the 64-min region of the Escherichia coli K-12 genome and contains no open reading frames. The purified CsrA-CsrB ribonucleoprotein complex was active in regulating glg (glycogen biosynthesis) gene expression in vitro, as was the RNA-free form of the CsrA protein. Overexpression of csrB enhanced glycogen accumulation in E. coli, a stationary phase process that is repressed by CsrA. Thus, CsrB RNA is a second component of the Csr system, which binds to CsrA and antagonizes its effects on gene expression. A model for regulatory interactions in Csr is presented, which also explains previous observations on the homologous system in Erwinia carotovora. A highly repeated nucleotide sequence located within predicted stem-loops and other single-stranded regions of CsrB, CAGGA(U/A/C)G, is a plausible CsrA-binding element.