Intraperitoneal (IP) injection of a fecal anorexigenic substance (FS-T) induced significant suppression of feeding and this suppression recovered on the second day. At 2 hr after IP injection, at the time of maximum feeding suppression, plasma glucose, insulin and free fatty acid (FFA) levels did not change but amino acid level decreased. Intra-third cerebral ventricle (ICV) infusion of FS-T induced parallel but more potent feeding suppression. Analysis of meal patterns demonstrated that suppression of feeding after ICV treatment continued into the second day. FS-T was applied electrophoretically to glucose-sensitive and non glucose-sensitive neurons in the lateral hypothalamic area (LHA) and to glucoreceptor and non glucoreceptor neurons in the ventromedial hypothalamic nucleus (VMH). It significantly inhibited glucose-sensitive neurons but not non glucose-sensitive neurons, and excited both neuron types in the VMH. FS-T might thus work directly through the hypothalamic feeding control centers to suppress feeding. Even after pronase treatment of FS-T, a non-dialysable fraction of large molecular weight, consisting of protein and carbohydrate, maintained the original anorexigenic activity.