In male rodents, complete reproductive function is critically dependent on adequate estrogen action at different levels of the hypothalamic-pituitary-testicular axis. However, administration of high doses of estrogen during the critical period of neonatal differentiation results in multiple defects in the reproductive axis that disrupt male fertility, the molecular mechanism(s) behind such a phenotype remaining poorly characterized. The aim of this study was twofold: (1) to characterize the effects of neonatal estrogenization upon the pattern of estrogen receptors ERα and ERβ mRNA expression in the pituitary and hypothalamus of the male rat, and (2) to evaluate the ability of estrogen to acutely regulate pituitary and hypothalamic ERα and ERβ mRNA expression in the male rat. To achieve the first goal, groups of male rats were treated on the first day of life with estradiol benzoate (EB; 500 µg/rat), and pituitary and hypothalamic expression of ERα and ERβ mRNA levels were assayed by semiquantitative RT-PCR at different ages from the neonatal period until adulthood (days 1–75 of life). In addition, the mechanism(s) of altered pituitary expression of ERα and ERβ messages in neonatally estrogenized rats was explored by comparison of the effects of neonatal treatment with estrogen or a potent gonadotropin-releasing hormone (GnRH) antagonist. For the second goal, the acute effects of a single dose of EB (12.5–25 µg/rat) on hypothalamic and pituitary ERα and ERβ mRNA expression were assessed in prepubertal and adult male rats. Neonatal estrogenization permanently decreased pituitary ERα and ERβ mRNA expression levels respective to control values at all ages studied. This pattern of response was similar to the short-term effects of neonatal blockade of endogenous GnRH actions. In contrast, neonatal exposure to estrogen transiently increased the hypothalamic expression levels of ERα and ERβ messages. This effect was detected in neonatal (5-day-old), infantile (15-day-old) and prepubertal (30-day-old) rats, and it disappeared at puberty. In addition, estrogen was able to acutely regulate mRNA expression levels of its cognate receptor subtypes in the pituitary and hypothalamus of intact male rats. In adult (75-day-old) males, EB administration (25 µg/rat) induced a significant time-dependent decrease in pituitary ERα and ERβ mRNA levels. In contrast hypothalamic expression of ERα and ERβ messages was increased after acute exposure to EB (12.5 µg/rat) in prepubertal males (30 days old); yet differences in the time course of the response to estrogen were noticed between targets. In conclusion, our data indicate that estrogen is able to neonatally imprint and acutely regulate mRNA expression levels of ERα and ERβ in the pituitary and hypothalamus of the male rat. Regulation of pituitary and hypothalamic ERα and ERβ gene expression by the cognate ligand represents a novel mechanism whereby estrogen modulates its own biological actions upon different levels of the male reproductive axis throughout the life span.