A plate assay was developed for the detection of anaerobic bacteria that produce azoreductases. With this plate assay, 10 strains of anaerobic bacteria capable of reducing azo dyes were isolated from human feces and identified as Eubacterium hadrum (2 strains), Eubacterium spp. (2 species), Clostridium clostridiiforme, a Butyrivibrio sp., a Bacteroides sp., Clostridium paraputrificum, Clostridium nexile, and a Clostridium sp. The average rate of reduction of Direct Blue 15 dye (a dimethoxybenzidine-based dye) in these strains ranged from 16 to 135 nmol of dye per min per mg of protein. The enzymes were inactivated by oxygen. In seven isolates, a flavin compound (riboflavin, flavin adenine dinucleotide, or flavin mononucleotide) was required for azoreductase activity. In the other three isolates and in Clostridium perfringens, no added flavin was required for activity. Nondenaturing polyacrylamide gel electrophoresis showed that each bacterium expressed only one azoreductase isozyme. At least three types of azoreductase enzyme were produced by the different isolates. All of the azoreductases were produced constitutively and released extracellularly.