Adrenocorticotropic hormone (ACTH) and melanophore-stimulating hormone (MSH) are produced in the pars distalis and pars intermedia, respectively, throughout vertebrates. These hormones together with beta-endorphin are encoded on a single gene proopiomelanocortin (POMC) in gnathostomes, but in the sea lamprey, an agnathan, ACTH and MSH are encoded on two separate genes, proopiocortin (POC) and proopiomelanotropin (POM), respectively. Moreover, the nucleotide sequences of 5'-flanking regions of the POC and POM genes are significantly different from each other. To investigate the potential promoter activities of the POC and POM genes, we constructed promoter reporter plasmids by fusing the 5' flanking sequences (nucleotides -1151 to +31 and -2510 to +51, respectively) to a firefly luciferase gene. Transient transfection studies in AtT-20/D16v cells, which derived from a mouse pituitary tumor cell line, revealed that the 5'-flanking sequence of the POC gene did not exhibit promoter activity, whereas that of the POM gene showed the activity at high levels nearly equivalent to SV40 promoter. Analysis of a series of the 5'-deleted reporter for the POM gene in the AtT-20/D16v cells demonstrated that the 422 bp 5'-flanking sequence was sufficient for promoter activity, while the sequence from -853 to -574 may contain negatively acting regulatory elements. Because the POC and POM genes are supposed to have differentiated from a common ancestor, during evolution, the POC gene may lack essential element(s) for expression in the AtT-20/D16v cells.