A cDNA clone encoding the soluble guanylyl cyclase alpha2 subunit was isolated from medaka fish (Oryzias latipes) and designated as OlGCS-alpha2. The OlGCS-alpha2 cDNA was 3,192 bp in length and the open reading frame (ORF) encodes a protein of 805 amino acids. The deduced amino acid sequence has high similarity to that of the mammalian alpha2 subunit gene except for the N-terminal regulatory domain. The C-terminal 5 amino acids, "RETSL", which have been reported to interact with the post synaptic density protein (PSD)-95 were conserved. An RNase protection assay with adult fish organs showed that OlGCS-alpha2 was expressed mainly in the brain and testis. The complete nucleotide sequence (about 41 kbp) of the OlGCS-alpha2 genomic DNA clone isolated from a medaka fish BAC library indicated that the OlGCS-alpha2 gene consisted of 9 exons and 8 introns. The 5'-flanking region and larger introns, such as introns 1, 4, and 7, contained the several fragments conserved in the nucleotide sequences of Rex6 (non-long terminal repeat retrotransposon), MHC class I genomic region, and OlGC1, the medaka fish homolog of the mammalian guanylyl cyclase B gene. Linkage analysis on the medaka fish chromosome demonstrated that the OlGCS-alpha2 gene was mapped to LG13; this mapping position was different from those for the OlGCS-alpha1 and OlGCS-beta1 genes (LG1).