Changes in mRNA expression for estrogen receptor (ERβ) in relation to mRNAs for LH receptor (LHr) and cytochrome P450 enzymes were examined in granulosa and theca cells from proestrous rat ovarian follicles. Of the 30 ovaries harvested from 15 adult rats, 24 were processed for in situ hybridization, and the remaining were used for reverse transcription-polymerase chain reaction. Messenger RNAs for ERβ, LHr, cytochrome P450 side-chain cleavage enzyme (P450_scc), 17α-hydroxylase (P450_c17), aromatase (P450_arom), and steroidogenic acute regulatory protein (StAR) were localized in cross sections of ovaries by in situ hybridization and quantified in granulosa and theca cell layers by a computer-image analyzing system. Ovarian follicles were classified as healthy or atretic. Healthy follicles were divided into four size groups: very small (40–100 μm), small (101–275 μm), medium (276–450 μm), and large (451–850 μm). Atretic follicles were divided into medium (276–450 μm) or large follicles (451–850 μm). A low level of ERβ mRNA expression was first detected in granulosa cells of very small healthy follicles, and the expression increased progressively up to medium-sized follicles. The expression of ERβ mRNA was highest (P < 0.01) in medium-sized follicles that was followed by a decrease (P < 0.01) in large follicles. Messenger RNAs for LHr, P450_scc, and P450_arom were first detected in granulosa cells of medium-sized healthy follicles, while mRNAs for LHr, P450_scc, P450_c17, and StAR were first detected in theca cells associated with very small follicles. The highest expression of LHr, P450_scc, P450_c17, P450_arom, and StAR was seen in granulosa and/or theca cells of large healthy follicles. In atretic follicles, level of gene expression was relatively low in both granulosa and theca cells. In conclusion, stage-specific expression of ERβ mRNA was observed in granulosa cells during follicular development. The increased expression of ERβ and a concomitant initiation of LHr, P450_scc, and P450_arom expression in granulosa cells of medium follicles may signify a role for estrogen in follicular development. Also, a strong correlation between ERβ mRNA expression in granulosa cells, and the expression of mRNAs for LHr, P450_scc, P450_c17, and StAR in theca cells associated with growing follicles suggests a possible role for estrogen in steroidogenesis.