The amino acid sequence of the histidine binding protein of Salmonella typhimurium was determined by automated sequence analysis of reduced and S-pyridylethylated histidine binding protein and fragments derived by chemical and enzymatic cleavage of the native protein. The fragments were the products of cleavage at methionine residues by cyanogen bromide, cleavage at tryptophan residues by 2-nitrophenylsulfenyl-3-methyl-3-bromo-3H-indole (BrNps-skatole), limited enzymatic digestion at arginine residues, and enzymatic digestion at Glu-X bonds by the Staphylococcus aureus V8 protease. The sequence of the COOH-terminal residues was determined using bovine carboxypeptidases A and B and amino acid analysis. The histidine binding protein was found to contain 238 amino acid residues and to have a molecular weight of 26,104 calculated from sequence.