Estrogen plays a key role in the regulation of many pituitary hormones. The presence of estrogen receptor-b (ERb) messenger RNA (mRNA) has been demonstrated in the adult anterior pituitary by RT-PCR to be at a level much greater than that of ERb mRNA. Because the number of ERs has been shown to change during devel- opment, in this study we examined the distribution of pituitary ERb mRNA in adult and prepubertal rats. Using RT-PCR, we confirmed that ERb mRNA expression is less than that of ERa mRNA in adult females. In contrast, in prepubertal female pituitaries, ERb mRNA levels are much greater than those of ERa mRNA. Film densitometric analysis of whole pituitaries, similarly showed that ERb mRNA is greater in prepubertal pituitaries than in adult pituitaries. However, after emulsion autoradiography, cell counts confirmed that prepu- bertal and adult pituitaries differ, not in the level of ERb mRNA expression, but in the number of cells expressing ERb mRNA. In postnatal day 15 pituitaries, there were twice as many cells per mm2 as in adults. A comparison between prepubertal males and females showed that females exhibited a 2-fold greater level of ERb mRNA expression. To determine which cell types express ERb mRNA, we performed in situ hybridization for ERb mRNA coupled with immu- nohistochemistry for FSH or PRL. In prepubertal pituitaries, 84.5 6 2.3% of FSH-immunoreactive cells also express ERb. Nearly all of the PRL-immunoreactive cells lacked ERb mRNA. These data demon- strate sex- and age-related differences in ERb mRNA expression in the anterior pituitary. Furthermore, these data suggest that ERb is not the specific mediator of estrogen action in lactotrophs, whereas ERb may be the prime mediator of estrogen action in FSH-containing gonadotrophs. (Endocrinology 139: 5151-5156, 1998)