Estradiol derived from neural aromatization of gonadal testosterone plays a key role in the perinatal organization of the neural circuitry underlying male sexual behavior. The aim of this study was to investigate the contribution of neural estrogen receptor (ER) β in estradiol-induced effects without interfering with its peripheral functions. For this purpose, male mice lacking ERβ in the nervous system were generated. Analyses of males in two consecutive tests with a time interval of two weeks showed an effect of experience, but not of genotype, on the latencies to the first mount, intromission, pelvic thrusting and ejaculation. Similarly, there was an effect of experience, but not of genotype, on the number of thrusts and mating length. Neural ERβ deletion had no effect on the ability of males to adopt a lordosis posture in response to male mounts, after castration and priming with estradiol and progesterone. Indeed, only low percentages of both genotypes exhibited a low lordosis quotient. It also did not affect their olfactory preference. Quantification of tyrosine hydroxylase- and kisspeptin-immunoreactive neurons in the preoptic area showed unaffected sexual dimorphism of both populations in mutants. By contrast, the number of androgen receptor- and ERα-immunoreactive cells was significantly increased in the bed nucleus of stria terminalis of mutant males.