Certain strains of viridans streptococci bind platelets, which release ATP from dense granules and then aggregate. By hydrolyzing the released ATP to the platelet agonist, ADP, cell wall-associated ATPase activity of Streptococcus sanguis may amplify the aggregation of platelets. To identify and characterize this ecto-ATPase activity, whole cells were incubated with [14C]-ATP. The cell-free nucleotides were separated by thin-layer chromatography and quantified by liquid scintillation counting. Whole-cell activity showed temperature and pH optima in the physiological range. To isolate a soluble fraction with ATPase activity from the cell wall, whole cells were digested under osmotically stable conditions to produce protoplasts. Protoplasts and cells were separated from soluble cell wall materials by centrifugation. ATPase activity in cell fractions was identified by zymograms of native 8% polyacrylamide gels after electrophoresis. The ecto-ATPase preparation, membrane and cytoplasmic ATPase in lysed protoplasts showed different zymograms and sensitivity to inhibition by DCCD, ouabain vanadate, azide and NEM. In electron micrographs of ultrathin sections of cells of S. sanguis, ATPase activity was localized to the cell wall. Since the pattern of localization to the wall changed with the phase of growth, the ecto-ATPase of S. sanguis may be associated with the development and maintenance of the cell wall.