Parathyroid hormone (PTH) regulates osteoblasts via a G protein-linked PTH/PTH-related protein (PTHrP) receptor. PTH effects on PTH/PTHrP receptor gene expression were studied in UMR 106 osteoblast-like cells. In heterogeneous nuclear RNA and Northern analysis, PTH suppressed PTH/PTHrP receptor transcription. We cloned the 7-kb promoter region of the rat PTH/PTHrP receptor gene and transiently transfected chimeric deletion constructs containing the 5 ' -flanking region and the luciferase gene into UMR 106 cells. In transfected cells the minimal region for basal promoter activity was between positions -128 and +103. The 5 ' -flanking region of exon U1 contained several putative-binding sites for Sp1 and the myc-associated zinc finger protein (MAZ). The minimal PTH-suppressive region (PTHSR) was between +1 and +25 in exon U1, but the 5 ' -flanking region or Sp1 and MAZ-binding sites also were required for PTH-mediated repression. By gel mobility shift assay PTH markedly decreased binding of PTHSR-protein complex in UMR 106 cells. The mutation experiments showed that the most critical sequence for the repression of PTH was 5 ' -GGGGGAGGGGAG-3 ' (+1 to +12) of PTHSR. This represents the first characterization of a PTH-suppressive region of the PTH/PTHrP receptor gene in rat.