The trimeric phosphocarrier protein factor III specific for galactosides was investigated by 1H NMR spectroscopy. The protomer contains four histidyl residues with acidic pK values in the range 5.6-6.2. One of the histidyl residues, His-B, carries the phosphoryl group. The pK value of His-B increases from 6.0 to 8.6 upon phosphorylation. To determine the position of the phosphoryl group with respect to the nitrogens required the isolation of a peptide T-2 containing the phosphorylated active-center histidine and one of the other histidines. The pK value and the chemical shift of the phosphopeptide clearly indicated the phosphorus to be bound to the N-3 atom of the imidazole ring. The temperature dependence of the factor III spectrum demonstrates multiple conformations which exchange rapidly on the NMR time scale. Titration of factor III with HPr protein showed an upfield shift of the active-center histidine, indicating complex formation between both proteins. Phosphorylation of both proteins abolished the interaction, which is plausible from mechanistic considerations.