A most probable number (MPN) method has been described for the enumeration of bacteria responsible for 2,4-dinitrophenol (DNP) degradation in activated sludge cultures. Because of the encapsulation of cells within the activated sludge floc, the method of pretreatment may be critical for producing a homogeneous distribution of bacteria to insure accurate dilution and enumeration. In these experiments, pretreatments using mechanical shear and incubation of cultures in EDTA have been tested. The combination of mechanical shearing in a high speed blender and incubation in a 0.5% EDTA solution resulted in significantly higher MPN values for DNP-degrading organisms growing in activated sludge in sequencing batch reactors (SBRs) which had been supplemented with glucose. Shearing of flocs and the combination of shearing and EDTA treatment produced the identical MPN values in both pure and activated sludge cultures grown only in DNP.