We cloned and characterized two subfamilies of olfatory receptor (OR) genes from medaka fish (Oryzias latipes). Southern blot analysis showed that each of the two subfamilies, designated as subfamilies Y and E, consists of about five members, as usually observed for other vertebrate ORs. Analyses of the genomic clones encoding these members revealed that two members of subfamily Y and four members of subfamily E are tandemly reiterated in 15 and 22 kbp regions of the medaka genome, respectively. The members of each subfamily show very similar amino acid sequences, with similarities greater than 70%. However, the similarities to the sequences of other vertebrate ORs are lower. Members of subfamily Y show amino acid sequence similarities of ca. 30% to other fish ORs, including subfamily E members, as well as to mammalian ORs. On the other hand, members of subfamily E show sequence similarities of ca. 50% to other fish ORs and ca. 30% to mammalian ORs. Phylogenic analyses of various fish ORs, including medaka, catfish, and zebrafish ORs, indicate that the primary structures of fish ORs are diverse compared with those of mammalian ORs, which consist of much larger numbers of members. The expression patterns of subfamilies Y and E genes in the olfactory epithelium of adult medaka fish were examined by in situ hybridization, showing that the frequency of positive signals is different between the two subfamilies: about 2% of the olfactory neurons are positive to probes for members of subfamily Y, while less than 1% are positive to probes for members of subfamily E. These results indicate that each subfamily is under different transcriptional control.