Nuclear hormone receptors (NRs) are ligand-inducible transcription factors that mediate critical functions in many species. The majority of novel NRs have hitherto been cloned from cDNA libraries by virtue of their homology to previously identified receptors. In this study, we validate a genomic DNA-based approach to isolating NRs by cloning the retinoic acid receptor-alpha (RARalpha) gene from the genome of the Japanese pufferfish, Fugu rubripes. The fRARalpha gene is more compact than its human and murine counterparts and demonstrates a highly conserved genomic organisation and amino acid sequence, generating two isoforms (fRARalpha1 and fRARalpha2) with divergent aminoterminal domains. In addition, a conserved regulatory element containing a retinoic acid response element was identified upstream of the fRARalpha2-specific exon, implying that retinoid induction of this isoform is evolutionarily conserved and critical to its function in vivo. We propose two uses for the Fugu genome in the study of NRs: the isolation of novel NRs that exhibit restricted spatio-temporal expression from genomic DNA and the identification of evolutionarily conserved promoter or intragenic regulatory DNA elements.