The driving force behind this new chromatographic technique was to develop a method for purifying preparative quantities of phosphohistidines in a single step that provided good resolution wit eluants that could be easily removed. The current method can provide milligram quantities of each phosphohistidine; however, the later 1H NMR analysis of the dried, individually purified phosphohistidines showed that histidine was present along with each of the individual phosphohistidines. The stability of each phosphohistidine during storage does not appear to be a problem because the amounts of histidine contamination of freshly freeze-dried samples were compared with those of samples stored at -80 degrees C under nitrogen for 2 weeks and were found to be similar (data not shown). Possibly, the lyophilization and preparation of solutions for NMR analysis resulted in a certain amount of hydrolysis of phosphohistidine, particularly with the less stable 1- and 1,3-forms (5, 6). It was noted that when the lyophilized samples were initially dissolved in D2O for NMR analysis, the pH was between 6 and 7; this may have resulted in some hydrolysis of the phosphohistidines. This hydrolysis can be reduced by the addition of 50 mM potassium hydroxide to the pooled fractions collected from the chromatography, so that the alkalinity of the samples is maintained throughout the subsequent processes. The data obtained for the assignment of individual phosphohistidines by 1H and 31P NMR analysis seem consistent with those obtained by other independent studies (6, 10). The NMR analysis was a powerful tool in assigning the identity of each purified phosphohistidine, although caution should be used when considering free phosphohistidines as references for NMR detection of phosphohistidines in native proteins. Lecroisey et al. (10) showed that there were differences between the chemical shifts observed for free phosphohistidine compared to those for phosphohistidine in dipeptides. However, for the purposes of phosphoamino acid analysis, these purified phosphohistidines are used by this group as references in the detection of phosphohistidine in proteins.