Glucose-adapted Streptococcus faecalis produced little if any (14)CO(2) from glucose-1-(14)C, although high levels of glucose-6-phosphate dehydrogenase (EC 188.8.131.52) and 6-phosphogluconate dehydrogenase (EC 184.108.40.206) were detected in cell-free extracts. Metabolism of glucose through the oxidative portion of the hexose-monophosphate pathway was shown to be regulated in this organism by the specific inhibitory interaction of the Embden-Meyerhof intermediate, fructose-1, 6-diphosphate (FDP), with 6-phosphogluconate dehydrogenase. Glucose-6-phosphate dehydrogenase activity was unaffected by FDP. The S. faecalis 6-phosphogluconate dehydrogenase was partially purified from crude extracts by standard fractionation procedures and certain kinetic parameters of the FDP-mediated inhibition were investigated. The negative effector was shown to cause a decrease in V(max) and an increase in the apparent K(m) for both 6-phosphogluconate and nicotinamide adenine dinucleotide phosphate (NADP). These effects were apparently a consequence of the ligand interacting with the enzyme at a site distinct from either the substrate or the coenzyme sites. Among the evidence supporting this was the fact that beta-mercaptoethanol blocked completely FDP inhibition, but had no effect on catalytic activity. The possibility that the regulation of 6-phosphogluconate dehydrogenase activity by FDP might be of some general significance was suggested by the observation that this enzyme from several other sources was also sensitive to FDP.