A novel membrane guanylyl cyclase (GC), OlGC9, was identified in the intestine of the medaka fish Oryzias latipes by the isolation of a full-length cDNA clone (3783 bp). Phylogenetic analysis indicated that OlGC9 belongs in the enterotoxin/guanylin receptor membrane GC subfamily. The nucleotide and deduced amino acid sequences of OlGC9 were highly homologous to those of OlGC6, another enterotoxin/guanylin receptor membrane GC in medaka fish. Linkage analysis of the medaka fish chromosome demonstrated that the OlGC9 gene was mapped to LG8, which distinguishes it from the OlGC6 gene. Determination of the cGMP concentrations in COS-7 cells expressed with OlGC9 indicated that Escherichia coli heat-stable enterotoxin (STa) stimulated the activity of OlGC9 in a concentration-dependent manner, although it did not activate the OlGC6 expressed in the COS-7 cells. The 5'-flanking region of the OlGC9 gene important for its transcription was partially determined using both CACO-2 cells and COS-1 cells, and was not found to be conserved with respect to either the mammalian GC-C gene or the OlGC6 gene.