Updated PubMed. For decades, extreme difficulties in analyzing histidine phosphorylation have limited the study of phosphohistidine signaling. Here we report a method revealing widespread and abundant protein histidine phosphorylation in E. coli. Our new approach generated the largest E. coli phosphoproteome dataset to date, of which a remarkable high percentage (~10%) are phosphohistidine sites. This resource enables a major step forward towards a better understanding of the biological function of histidine phosphorylation.