Reversible phosphorylation of proteins is a vital regulatory mechanism that touches virtually every aspect of cellular biology. The overwhelming majority of studies in higher eukaryotes have focused on phosphorylation of just 3 amino acid residues: serine, threonine and tyrosine. While phosphorylation of these residues is undoubtedly of great importance, recent evidence suggests that non-canonical protein histidine phosphorylation, which is widespread in bacteria, may be of equal importance in eukaryotes. Study of histidine phosphorylation has lagged not because of a lack of intrinsic importance, but due to the extreme heat and acid lability of the phosphohistidine (pHis) phosphoramidate (P-N) bond which precludes the use of standard protein enrichment and analytical methods. However, recent advances in the development of reliable pHis antibodies for immunodetection and immunoprecipitation, coupled with ongoing advances in mass spectrometric methods and analysis, present a window of opportunity for making substantial and rapid progress in this area. We are well poised to seize this opportunity. We have published in the field and have in-house experience working on histidine phosphorylation. Our preliminary results show that pHis may be relevant to other core areas of interest in our laboratory, namely cancer and metabolism. We propose to clarify this potential link using cell and animal models, with the final aim of establishing clinical relevance. We also propose to identify novel substrates of the two known mammalian histidine protein kinases (NME1 and NME2), to add to what is only a handful of currently known histidine-phosphorylated proteins.