The human growth hormone (GH) family consists of five genes, including the placental chorionic somatomammotropins (CS), within a single locus on chromosome 17. Based on nuclease sensitivity, the entire GH/CS locus is accessible in pituitary chromatin, yet only GH-N is expressed. Previously, we reported a P sequence element (263P) capable of repressing placental CS-A promoter activity in transfected pituitary (GC) cells, and our data indicated a possible role for nuclear factor-1 (NF-1) and regulatory factor X1 (RFX1) in this repression. Here we show the formation of two independent pituitary complexes in vitro: a repressor complex containing NF-1, and a non-functional complex containing RFX1. In vitro repressor function is stabilized by the presence of P sequence element (PSE)-C, downstream of the previously characterized PSE-A and PSE-B. Repressor function is also dependent on an intact Pit-1 binding site in the CS-A promoter. Electrophoretic mobility shift assays with PSE-C reveal binding of the hepatocyte nuclear factor 3/forkhead (HNF-3/fkh) family of transcription factors in rat pituitary GC cells. This observation is extended to human pituitary tissue, where HNF-3 association with P sequences is confirmed by chromatin immunoprecipitation. Furthermore, protein-protein interactions between HNF-3 and NF-1 family members are demonstrated. These results identify HNF-3 as an additional member of the pituitary P sequence regulatory complex, implicating it in tissue-specific expression of the human GH/CS family.