Metabolomics and systems biology require the acquisition of reproducible, robust, reliable, and homogeneous biological data sets. Therefore, we developed and validated standard operating procedures (SOPs) for quenching and efficient extraction of metabolites from Escherichia coli to determine the best methods to approach global analysis of the metabolome. E. coli was grown in chemostat culture so that cellular metabolism could be held in reproducible, steady-state conditions under a range of precisely defined growth conditions, thus enabling sufficient replication of samples. The metabolome profiles were generated using gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS). We employed univari-ate and multivariate statistical analyses to determine the most suitable method. This investigation indicates that 60% cold (-48 °C) methanol solution is the most appropriate method to quench metabolism, and we recommend 100% methanol, also at-48 °C, with multiple freeze-thaw cycles for the extraction of metabolites. However, complementary extractions would be necessary for coverage of the entire complement of metabolites as detected by GC/TOF-MS. Finally, the observation that metabolite leakage was significant and measurable whichever quenching method is used indicates that methods should be incorporated into the experiment to facilitate the accurate quantification of intracellular metabolites. Investigations in microbial metabolomics require reliable and reproducible analysis of the metabolites in the cell across a broad dynamic range of concentrations (nanomolar to millimolar) and from a range of different chemical functionalities. The development of robust and reliable experimental protocols for all steps in the experimental procedure, ranging from biomass cultivation, quenching , and extraction of the metabolome to the quantitative analysis of the metabolites, is required. The analytical methodology available for the detection and identification of metabolites is relatively well developed (see refs 1-4), and these platforms have been optimized. 5,6 More recently the Metabolomics Standards Initiative (refs 7-9 and see http://msi-workgroups.sourcefor-ge.net/) as well as others 3,10-12 have started to discuss the standardization of metabolomics experiments from biological growth-collection through chemical analysis to data processing. However, less consideration has been directed toward optimizing the methods for arresting metabolic activity by quenching protocols and the extraction methods for intracellular metabolites. Villas-Boas et al. have developed optimized quenching and extraction methods for the yeast Saccharomyces cerevisiae. However , there are significant chemical and physical differences between the cell envelopes of eukaryotic and prokaryotic organisms , and this means that these protocols may not be directly transferable to prokaryotic organisms. Most notably, the leakage of metabolites during quenching with organic solvents in eukary-otic …