Five members of the human growth hormone (GH) gene family are located at a single locus on chromosome 17. Growth hormone is expressed in the pituitary under the control of the tissue-specific factor Pit 1/GHF-1, and chorionic somatomammotropin (CS) -A, -B, and -L, as well as placental GH variant, are expressed specifically in the placental syncytiotrophoblast. Despite this specificity in vivo, the CS-A promoter can bind Pit 1/GHF-1 and allow CS-A promoter activity in pituitary tumor cells after gene transfer. We have identified and characterized PSF sequences associated with only the placental members in the GH/CS locus which repress placental promoter activity > 90% in transfected pituitary cells. These sequences do not significantly affect promoter function in placental cells after gene transfer. Repressor activity correlates with binding of protein at two sites (PSF-A and PSF-B) with pituitary, but not placental, nuclear extracts. Competition studies suggest an interaction between PSF and Pit 1/GHF-1 proteins. These results indicate that PSF protein can repress CS-A promoter activity in a tissue-specific manner in vitro and provide a possible mechanism by which expression of placental members of the GH family are inhibited in the pituitary in vivo.