A global methylation-based technique was used to identify, display, and quantitate the in vivo occupancy of numerous protein-binding sites within the Escherichia coli genome. The protein occupancy profiles of these sites showed variation across different growth conditions and genetic backgrounds. Of the 25 sites identified in this study, 24 occurred within 5' noncoding regions. Protein occupancy at 13 of these sites was supported by independent biochemical and genetic evidence. Most of the remaining 12 sites fell upstream of genes with no previously known function. A multivariate statistical analysis was utilized to group such uncharacterized genes with well-characterized ones, providing insights into their function based on a common pattern of transcriptional regulation.