The ontogeny of substance P-containing neuron system in the forebrain and upper brain stem of the rat was investigated by means of the indirect immunofluorescence technique. Substance P-positive structures first appeared in the primordium of the epithalamus and the area which surrounded the commissura posterior of the rat fetus corresponding to gestational day 14 (10-12 mm embryos). On and after gestational day 14, substance P-positive structures gradually made their appearance in various areas of the forebrain and upper brain stem. Substance P-positive structures thus continued to increase in number and in density during the fetus and perinatal stage and showed histochemically maximum content at the stage between postnatal days 5 and 15. After then, substance P-positive neurons tended to decrease in number as the rats grew, while substance P-positive fibers maintained in general their strong immunoreactivity even in the adult rats. The present study demonstrates that substance P-positive structures appear at a very early ontogenetical stage. This suggests that substance P might play an important role in the development of the forebrain and upper brain stem in addition to its neurotransmitter or neuromodulator functions.