Abstract The occurrence of a pyrophosphate:l-serine phosphotransferase in extracts of Propionibacterium shermanii is reported. A 102-fold purification, with 15% recovery, was obtained by successive use of protamine sulfate precipitation, acidification, ammonium sulfate precipitation, and chromatography on DEAE-cellulose and on Bio-Gel P-150 columns. This purification eliminates inorganic pyrophosphatase and serine phosphatase activity present in the crude extract, but a serine-serine phosphate exchange activity remains. Evidence is presented that this exchange is catalyzed by a second enzyme. The enzyme is stable at room temperature, but rapidly loses activity above 50°. In the presence of l-serine the enzyme exhibits a somewhat higher lability to preliminary incubation at elevated temperatures. The Km for PPi is 1.0 x 10-4 m; the Km for l-serine is 1.9 x 10-3 m. The enzyme is specific for both substrates. ATP is not a substrate. Tripolyphosphate can substitute to some extent for PPi. d-Serine and l-cysteine are not phosphorylated. dl-Homoserine is phosphorylated to a slight extent, as is l-threonine. Mg++ greatly stimulates the reaction. Some stimulation is obtained with Mn++ and with Co++. The Km for Mg++ at 1 mm PPi is 1.3 x 10-4 m, very close to the Km for PPi at 1 mm Mg++. Kinetic evidence suggests that the enzyme operates by a random equilibrium mechanism, involving a ternary complex of enzyme and substrates. The reaction has an apparent equilibrium constant of 480 at pH 6.7 and of 950 at pH 7.7, and an Arrhenius activation energy of 14.7 kcal per mole at pH 7.8.