Histidine-containing protein (HPr) of gram-positive bacteria was found to be phosphorylated at a seryl residue (P-ser-HPr) in an ATP-dependent reaction catalyzed by a protein kinase (J. Deutscher and M. H. Saier, Jr., Proc. Natl. Acad. Sci. U.S.A. 80:6790-6794, 1983). Here we describe the purification and characterization of a soluble enzyme of Streptococcus faecalis which splits the phosphoryl bond in P-ser-HPr. The enzyme has a molecular weight of ca. 7.5 X 10(4), as determined by its migration behavior on a Sephacryl S-200 column. On native polyacrylamide gels the purified enzyme produced only one protein band. On sodium dodecyl sulfate-polyacrylamide gels we found one major protein band of molecular weight 2.9 X 10(4) and two minor protein bands of molecular weights 2.3 X 10(4) and 7 X 10(4). Fructose 1,6-diphosphate, which stimulated the ATP-dependent, protein kinase-catalyzed phosphorylation of HPr, had no effect on the phosphatase activity. Other glycolytic intermediates also had no effect. However, inorganic phosphate, which inhibited the ATP-dependent HPr kinase, stimulated the P-ser-HPr phosphatase. EDTA at a concentration of 0.1 mM completely inhibited the phosphatase. Divalent cations like Mg2+, Mn2+, and Co2+ overcame the inhibition by EDTA. Fe2+, Zn2+, and Cu2+ had no effect, whereas Ca2+ slightly inhibited the phosphatase. ATP was also found to inhibit the phosphatase. Under conditions in which ATP severely inhibited the phosphatase, ADP was found to have no effect on the enzyme activity. Besides P-ser-HPr of S. faecalis, the phosphatase was also able to hydrolyze the phosphoryl bond in P-ser-HPr of Streptococcus lactis, Staphylococcus aureus, Bacillus subtilis, Streptococcus pyogenes, and Lactobacillus casei. Phosphoenolpyruvate-dependent o-nitrophenyl-beta-D-galactopyranoside phosphorylation, catalyzed by the S. aureus phosphoenolpyruvate:lactose phosphotransferase system, was about 150-fold decreased in the presence of P-ser-HPr of S. aureus, as compared with HPr. However, when P-ser-HPr was first incubated with P-ser-HPr phosphatase to allow complete hydrolysis of the phosphoryl bond, it had the same activity as HPr. Besides this cytoplasmic phosphoprotein phosphatase, we detected a membrane-bound phosphatase which also hydrolyzed the phosphoryl bond in P-ser-HPr.