AbstractAn essential part ofin vivoINT‐dehydrogenase assay is quantitation of the intensely colored, water‐insoluble iodonitrotetrazolium formazan (INTF) that forms intracellularly as the product of INT reduction. This procedure involves extraction of INTF from microbial cells by an organic solvent, permitting colorimetric measurement of the formazan. In the present study, it was found that dimethylsulfoxide (DMSO) provided the most consistent extraction of INTF from activated sludge and filamentous bacteria. The relative efficiency of 2 + 3 tetrachloroethylene/acetone (TA) was slightly greater than that of DMSO in some cases. However, this solvent only partially extracted INTF from one of the filament types tested (type 1701) and its performance was impaired by adsorption of ferrous ion to sludge. Methanol was generally the least efficient of the solvents tested. It was particularly ineffective in extracting INTF from Fe2+‐treated sludges. Permeabilization of cells with lysozyme or Triton X‐100 improved the INTF yields obtained with methanol. Triton X‐100,the more effective of the two permeabilizing agents, increased the INTF yield obtained with methanol to the same level as that obtained with D