Reverse transcription-polymerase chain reaction (RT-PCR) with specific GnRH cDNA primers performed on RNA from Rathke's pouches removed from pregnant rats at day 12 of gestation (e12) generated an amplified DNA fragment of the expected length (357 bp). The fragment hybridized with a labeled GnRH cDNA probe in Southern blotting. DNA sequencing demonstrated identity with the known nucleotide sequence of the corresponding segment of rat GnRH cDNA. To determine whether GnRH mRNA was located in the Rathke's pouch cells or in remnants of surrounding tissue not completely removed during preparation, the pouches were treated with collagenase. Based on light and electron microscopic examination, this treatment disconnected virtually all contaminating tissue, allowing the 'pure' Rathke's pouches to be picked-up separately. Again, RT-PCR generated a DNA fragment of the expected length, the fragment hybridized with the GnRH cDNA probe and showed the nucleotide sequence of the corresponding region of rat GnRH cDNA. In Rathke's pouches established in explant culture on e12, lactotrophs were well developed when examined 9 days later by immunostaining of prolactin in paraffin-embedded sections of the tissue. Computerized image analysis showed prolactin immunoreactivity in 8.0+/-1.1% of the section area. Addition of the potent and long-acting GnRH antagonist ORG 30276 to the crude preparation of Rathke's pouches caused a significant decrease in the relative area staining for prolactin. The latter effect was abolished by concomitant addition of GnRH. In preparations of pure Rathke's pouches (collagenase-treated), ORG 30276 failed to affect the relative area of prolactin immunoreactivity. GnRH mRNA remained expressed in explants of both crude and pure Rathke's pouches until the end of the culture period. It is concluded that the GnRH gene is expressed in Rathke's pouch as early as e12 and that GnRH may be a physiological paracrine/autocrine peptide stimulating the development of lactotrophs. Mesenchymal and/or neural factors may be essential for the latter system to function.