Advances in genome sequencing have revolutionized biology by providing the molecular blueprints for thousands of living organisms. Yet, the functions of a large fraction, as much as one-half, of the component parts remain unknown even for the best understood organisms, including Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae. Here, we describe our development of comprehensive genomic resources (ORFeome clone sets and mutant libraries) for systematic functional analysis of E. coli, summaries on our use of these resources, the GenoBase information resource for handling high-throughput experimental data obtained with them, and our creation of user workspaces at our Protein Function Elucidation Team ( http://www.PrFEcT.org ) website.