Neuroanatomical attempts have been made to determine the synapses between luteinizing hormone-releasing hormone (LHRH)-containing neurons and substance P (SP)-containing neurons in the hypothalamus of female rats. Wheat germ agglutinin was injected into the septopreoptic area (SPA) and found to be incorporated into certain SP-containing neurons within the arcuate nucleus and the ventrolateral portion of the anterior hypothalamus. Hence, we used a preembedding double immunostaining technique in demonstrating LHRH and SP neurons in the SPA. In light-microscopic preparations LHRH was labeled with 3,3’-diaminobenzidine tetrahydrochloride (DAB) as chromogen while SP was labeled with silver-gold particles; brown LHRH cells appeared to be surrounded by black silver-gold dots. In electron-microscopic preparations, the labelings for LHRH and SP were made reversely; SP was localized with DAB chromogen, and SP-containing axonal terminals appeared to make synaptic contacts on silver-gold-labeled LHRH cell bodies and dendritic processes. The terminals contained numerous small clear vesicles and some large dense-cored vesicles, and the synaptic membrane specialization appeared to be symmetric and asymmetric. These findings indicate that certain SP neurons existing in the arcuate nucleus and the ventrolateral portion of the anterior hypothalamus may project fibers to make synaptic contact with LHRH neurons in the SPA in the rat.