We have isolated and sequenced a cosmid clone from the compact genome of the Japanese pufferfish (Fugu rubripes) containing portions of three genes that have the same order as in human. The gene order is microtubule-associated protein (MAP-2), myosin light chain (MYL-1), and carbamoyl phosphate synthetase (CPS III). The intron-exon organization of Fugu CPS III is identical with that of rat CPS I, although the equivalent genomic fragments of rat and Fugu CPS span 87.9 and 21 kb, respectively. This is the first report of a piscine CPS III genomic structure and predicts a close evolutionary link between CPS III and CPS I. The 8-kb intergenic region between MYL-1 and CPS gave no clear areas of transcription factor-binding sites by pairwise comparison with shark or rat CPS promoter regions. However, there was a match with the rat myosin light chain 2 (MLC-2) gene promoter and a MyoD transcription factor-binding site 874 bp upstream of the MYL-1 gene.