Neurons in the lateral septum (LS) with projecting axons to the midbrain central gray (MCG) exert an inhibitory influence on lordosis. The number of such neurons is greater in female than in male rats. In this experiment, effects of neonatal estrogen on the density of the LS-MCG connections and on lordosis behavior were examined in female rats. On postnatal day 4 (day 0 = day of birth), females were injected subcutaneously with 50 or 100 µg estradiol benzoate (EB) or oil. On postnatal day 60, females and control males were gonadectomized. Behavioral tests were carried out after the implantation of silicone tubes containing estradiol. Lordotic activities in both males and EB-treated females were lower than in oil-treated females. After completing the behavioral tests, the animals were injected with Fluoro-Gold (FG), a retrograde tracer, into the right-side MCG and the number of FG-labeled neurons in the LS was measured. In all groups, the right-side LS ipsilateral to the FG injection had more FG-labeled neurons than the left-side LS. The number of FG-labeled neurons in the LS of oil-treated females was larger than that of males on both right and left sides. In the females treated with 100 µg EB (EB100), the number of FG-labeled neurons was comparable with that of males and lower than that of oil-treated females. The number of FG-labeled neurons in the EB50 females was also lower than that in oil-treated females, but tended to be larger than that observed in the EB100 group. These results indicate that neonatal estrogen decreases both lordotic activity and the density of the LS-MCG neural connections in female rats.