The Mn(2+)-oxidizing bacterium Pseudomonas fluorescens GB-1 deposits Mn oxide around the cell. During growth of a culture, the Mn(2+)-oxidizing activity of the cells first appeared in the early stationary growth phase. It depended on the O2 concentration in the culture during the late logarithmic growth phase. Maximal activity was observed at an oxygen concentration of 26% saturation. The activity could be recovered in cell extracts and was proportional to the protein concentration in the cell extracts. The specific activity was increased 125-fold by ammonium sulfate precipitation followed by reversed-phase and gel filtration column chromatographies. The activity of the partly purified Mn(2+)-oxidizing preparation had a pH optimum of circa 7 and a temperature optimum of 35 degrees C and was lost by heating. The Mn(2+)-oxidizing activity was sensitive to NaN3 and HgCl2. It was inhibited by KCN, EDTA, Tris, and o-phenanthroline. Although most data indicated the involvement of protein in Mn2+ oxidation, the activity was slightly stimulated by sodium dodecyl sulfate at a low concentration and by treatment with pronase and V8 protease. By polyacrylamide gel electrophoresis, two Mn(2+)-oxidizing factors with estimated molecular weights of 180,000 and 250,000 were detected.