Histidine-containing protein, HPr, is a phosphocarrier protein that is part of the bacterial phosphoenolpyruvate:sugar phosphotransferase system. HPr is phosphorylated by enzyme I, and P-HPr transfers the phosphoryl group to the IIA domain of a number of sugar-specific enzyme II complexes. Autocatalytic phosphoryl transfer between P-HPr and HPr has recently been reported [van Dijk, A. A., Eisermann, R., Hengstenberg, W., & Robillard, G. T. (1991) Biochemistry 30, 2876-2882]. Our results show that this phosphoryl transfer is due to an unidentified contaminant of HPr preparations. The phosphoryl transfer activity is not present in all HPr preparations. When present, the phosphoryl transfer activity can be removed by further purification or destroyed over time by resuspension of HPr preparations in water. There is no autocatalytic phosphoryl transfer between P-HPr and HPr.