Evolutionary studies of complement factor B (Bf) and C2 in lower vertebrates have revealed the presence of the Bf/C2 common ancestor-like molecule in lamprey (cyclostome) and the Bf molecule encoded by the duplicated genes closely linked to the major histocompatibility complex (MHC) in Xenopus (amphibian). To further define when Bf/C2 gene duplication occurred and when linkage between the Bf/C2 gene and the MHC was established, we amplified the Bf/C2 sequences in teleost, the Japanese medaka (Oryzias latipes), by reverse transcription - polymerase chain reaction with primers corresponding to the common amino acid sequences shared by mammalian Bf and C2. Only a single molecular species has been amplified, and the corresponding cDNA clones were isolated from the liver cDNA library. The longest insert contained 2384 nucleotides with an open reading frame of 754 residues. The deduced amino acid sequence showed 33.6% and 34.1% overall identity with the human Bf and C2 sequences, respectively, hence this clone was named medaka Bf/C2. The single-copy medaka Bf/C2 gene had exactly the same exon-intron organization as the mammalian Bf and C2 genes, and spanned about 8 kilobases. The Bf/C2 locus was mapped to the close proximity (2.9 cM) of the superoxide dismutase locus on the linkage group XX by the use of a restriction site polymorphism between two inbred strains of the medaka.