To verify the anatomical and functional connection of the lateral septum (LS) and periaqueductal gray (PAG) in inhibiting female sexual behavior, lordosis, in male rats, retrograde (Fluoro-Gold, FG) or anterograde (Phaseolus vulgaris-leucoagglutinin, PHA-L) tracer was injected into the PAG or LS on the right side, respectively, and FG-labeled cells or PHA-L-labeled axons in the forebrain and mesencephalon were determined in estrogen-treated castrated male rats. A ventral cut (VC) of the septum and a behavioral test were also conducted in some FG-injected rats. Furthermore, lordosis behavior was observed after chemical destruction of the septum by ibotenate. As a result, the lordosis quotient (LQ) in VC males was higher than that in control males without VC. FG-labeled neuronal cell bodies were found in the ipsilateral intermediate part of the LS in the control males but not in this area of the VC males. When neuronal cells in the intermediate part of the bilateral LS were completely destroyed by ibotenate, the LQ was higher than that in sham-lesioned male rats. These results suggest that a direct neural connection of the intermediate LS to the PAG has an inhibitory role in regulating lordosis in male rats. In addition, neuronal cell bodies in the intermediate LS exert an inhibitory influence. In the PHA-L experiment, labeled axons were seen in the ventral part of the LS, the medial forebrain bundle at the chiasmatic level, the lateral hypothalamus, the median region of the mesencephalon, and the rostral PAG in the side ipsilateral to the tracer injection site of the LS. Thus, these areas are thought to be involved in the pathway for lordosis-inhibition from the intermediate part of the LS to the PAG in male rats.