The present studies were designed to evaluate the role of substance P (SP) in the control of the release of luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL). SP was microinjected into the medial preoptic area (MPOA) of conscious, freely moving intact or orchidectomized (ORCX; 21 days post-ORCX) adult male rats. Microinjection of SP into the MPOA induced a significant decrease in plasma LH and FSH concentrations, effects which were accompanied by an elevation in plasma PRL concentration. To examine the participation of endogenously secreted SP in the activity of the MPOA neurons controlling release of these cited pituitary hormones, another study was performed in which either a potent and specific antagonist to SP (D-Pro2,D-Trip7,9-SP; SP-ANT) or an antibody against SP (SP-AB), was injected into the MPOA. SP-ANTand SP-AB both elevated plasma LH, FSH and decreased plasma PRL concentration. These data suggest that endogenous SP within the MPOA exerts an important inhibitory tonus over LH and FSH release and an excitatory tonus over PRL release. In conclusion, SP seems to participate as a neurotransmitter or neuromodulater in the control of LH, FSH and PRL secretion, at least in part, by acting at the level of MPOA, a region in which the neuronal cell bodies that produce LH-releasing hormone and the associated gonadotropin-releasing hormone-associated peptide are located.