The influence of dietary fructooligosaccharide (neosugar) on the fecal flora and activities of reductive enzymes was studied in 12 healthy, adult human subjects fed a controlled diet for 42 d and given 4 g neosugar/d between days 7 and 32. Fecal samples were collected before, during, and after supplementation with neosugar to enumerate total anaerobes, aerobes, bifidobacteria, and enterobacteria, and to assay for beta-glucuronidase, nitroreductase, and glycocholic acid hydroxylase. Although the controlled diet caused an increase in total anaerobes and bifidobacteria, the highest densities occurred during supplementation with neosugar. Total aerobes and enterobacteria were less affected by diet and neosugar. Neosugar caused beta-glucuronidase and glycocholic acid hydroxylase activities to decrease 75% and 90%, respectively; both increased after supplementation with neosugar was stopped. Nitroreductase activity declined 80% after the control diet was started, but was not affected by neosugar. These findings indicate that 4 g neosugar/d alters the fecal flora in a manner perceived as beneficial by decreasing activities of some reductive enzymes.