A redox dye-staining method using 5-cyano 2,3-ditolyl tetrazolium chloride (CTC) was modified for differential detection of metabolically active gram-positive and gram-negative bacteria. Exponentially growing cells of representative species of the phyla Actinobacteria, Firmicutes and Proteobacteria were stained with CTC in the presence of a substrate mixture, Meldola's Blue and KCN, and post-treated with acetone. Epifluorescence microscopy and spectrophotometric experiments showed that the acetone treatment resulted in the almost complete extraction of CTC formazan crystals from the gram-negative species, whereas it had little or no effect on CTC-stained gram-positive bacteria. Metabolically active gram-positive and gram-negative bacteria in municipal-sewage activated sludge measured by the modified CTC-staining method with microscopic detection composed 24 and 29% of the total count on average, respectively, both of which corresponded to approximately 74% of the viable gram-positive and gram-negative counts detected with a ViaGram Red⁺ Bacterial Gram Stain and Viability kit. Flow cytometric analyses of the CTC-stained and acetone-extracted activated sludge samples gave similar counts to those measured by epifluorescence microscopy. These results indicate that the modified CTC stain procedure with epifluorescence microscopy or flow cytometry can be used as a simple and rapid method for the estimation of metabolically active populations of gram-positive bacteria and gram-negative bacteria in the environment.