New denitrifying strains of phototrophic bacteria isolated from photosynthetic sludge reactors for wastewater treatment were characterized. All of the new isolates were mesophilic, nonhalophilic, facultative photoheterotrophs that were able to grow by anaerobic photosynthesis, aerobic respiration, or nitrate respiration. They had ovoid cells that were motile by single polar flagella, formed vesicular photosynthetic membranes together with bacteriochlorophyll a and carotenoids of the spheroidene series, required biotin, thiamine, and biotin as growth factors, and utilized a wide variety of organic compounds as electron donor and carbon sources. In these respects, the isolates most closely resembled Rhodobacter sphaeroides. However, they differed from this species in utilizing malonate and dulcitol but not tartrate as carbon sources and in their inability to grow anaerobically in darkness with trimethylamine N-oxide or dimethylsulfoxide as a terminal oxidant. Partial sequencing of 16S rRNA genes provided evidence for genetic differences between the new isolates and R. sphaeroides or other members of the genus Rhodobacter. Activities of nitrate reductase, nitrite reductase, and nitrous oxide reductase were detected in intact cells of one of the new isolates. All these enzyme activities were induced by cultivation with nitrate.