We have identified and characterized a pair of negative regulatory elements far upstream of the transcription start site of the human parathyroid hormone (hPTH) gene. Transfection of various types of cultured cells with a fusion plasmid containing 4.7 kilobase pairs of the 5'-flanking portion of the hPTH gene linked to the chloramphenicol acetyltransferase (CAT)-coding sequence generated only 10% of the CAT activity of a plasmid containing 684 base pairs of the 5'-flanking region of the hPTH gene. Deletion analyses reveal that there are at least two separate upstream DNA elements in the hPTH gene responsible for the negative regulation. We find that these cultured cells possess nuclear factors which specifically bind to several short DNA sequences within these elements and that these sequences can suppress transcription of the hPTH gene.