Protein-protein interactions play key roles in protein function and the structural organization of a cell. A thorough description of these interactions should facilitate elucidation of cellular activities, targeted-drug design, and whole cell engineering. A large-scale comprehensive pull-down assay was performed using a His-tagged Escherichia coli ORF clone library. Of 4339 bait proteins tested, partners were found for 2667, including 779 of unknown function. Proteins copurifying with hexahistidine-tagged baits on a Ni2+-NTA column were identified by MALDI-TOF MS (matrix-assisted laser desorption ionization time of flight mass spectrometry). An extended analysis of these interacting networks by bioinformatics and experimentation should provide new insights and novel strategies for E. coli systems biology.