Species-specific differences in genes encoding type II GnRH receptor indicate that a functional hepta-helical receptor is produced in monkeys but not in rodents, cows, chimpanzees, or humans. To further investigate the extent of evolutionary differences, we sequenced the type II GnRH receptor gene from wild-type Soay sheep. The gene was isolated by long-distance PCR using primers to PEX11beta and RBM8A genes known to flank type II GnRH receptor gene homologues. The gene spans 5.7-kb DNA and was sequenced after shot-gun subcloning. Its novel features include absence of a Pit-1 transcription factor binding site, a premature stop codon (TAG) in exon 1, an in-frame deletion of 51 bp (17 codons) in exon 2, and several nonconservative codon changes. Sheep breed variation in the gene was assessed using genomic DNA in PCR-restriction digest assays for the premature stop codon and in a PCR assay for the deletion. Both characteristics were present in all 15 breeds tested. Receptor gene expression was investigated using poly-A(+) RNA Northern analysis, RT-PCR, and in situ hybridization. An oligonucleotide probe to exon 1 revealed an alternative transcript in testis but not in pituitary gland. No transcripts in testis or pituitary were detectable using an exon 2-3 probe. All tissues examined including multiple brain areas and gonadotrope-enriched cell cultures were negative for type II GnRH receptor in RT-PCR. Testis and pituitary sections were negative with exon 1 riboprobes and exon 1 or 2-3 oligonucleotide probes in in situ hybridization. A hepta-helical type II GnRH receptor is therefore not expressed from this sheep gene.