The NtrC transcription factor is a member of a family of homologous prokaryotic regulatory proteins that participate in the transduction of extracellular and nutritional signals. It has been demonstrated that the phosphate group from a histidine residue of the phosphorylated NtrB protein autokinase is transferred to the NtrC protein. Phosphorylation of the NtrC protein is transient and activates its transcriptional enhancement activity. We have investigated the site of phosphorylation of the Salmonella typhimurium NtrC protein and find that it is an aspartate residue (Asp-54) that is found within a sequence conserved in all of the members of the family of regulatory proteins. We propose that this phosphorylation is an NtrC protein histidine phosphatase catalytic intermediate. This conclusion suggests that the NtrC family should be viewed not as kinase substrates but as enzymes that can catalyze the hydrolysis of their activated forms in a concentration-independent fashion. They are similar in this sense to eukaryotic signal-transducing GTPases.