The preparation of a novel phosphorus species, thiophosphoramidate, has enabled the specific thiophosphorylation of histidine at its 3-position. The rates of phosphorylation and thiophosphorylation of histidine are reported, as well as the spectroscopic properties of both thiophosphoramidate and 3-thiophosphohistidine. Structural assignment of the latter was made by analogy to the NMR properties of the known 3-phosphohistidine. The alkylation of 3-thiophosphohistidine by phenacyl bromide serves as a model for the introduction of labeling or probe reagents into histidine phosphorothioate-containing proteins.