A method is described for analysis of the intracellular concentrations of metabolic intermediates of the Embden-Meyerhof-Parnas pathway, the Entner-Doudoroff pathway, the pentose phosphate pathway, and the tricarboxylic acid cycle in cell pool extracts of Escherichia coli. A single anion-exchange HPLC run of 40 min allowed resolution of 27 anionic metabolite standards. Detection limits of 0.1 nmol per injection were achieved by use of a conductivity detector equipped with an anion self-regenerating suppressor and a uv detector. A boiling water extraction procedure was used to prepare cell pool extracts. Cochromatography of cell pool extracts and metabolite standards was used to confirm the identities of metabolites in the cell pool. As many as 16 metabolites could be detected and quantified in the cell pool extracts by using the described HPLC method. An analysis of metabolite concentrations in E. coli showed the dynamics of glucose metabolism during a 2-min transition from starvation to steady-state metabolism following addition of glucose. The ease and power of this method suggests general utility for in vivo metabolite analysis in a variety of experimental systems.