Abstract--The type B natriuretic peptide receptor (NPR-B) is the cognate receptor for the C-type natriuretic peptide and, as such, is responsible for signaling growth-suppressant activity in vascular smooth muscle cells. Here we report the isolation and characterization of the human (h) NPR-B gene promoter. Using 5' rapid amplification of cDNA ends analysis, we have identified the 5' terminus of the hNPR-B gene transcript 732 base pairs upstream from the presumed translation start site of the protein. We generated a series of 5' deletion mutants linked to a luciferase reporter and introduced these constructs into rat aortic smooth muscle cells or neonatal rat cardiac fibroblasts. Maximal expression was seen with a construct harboring 441 base pairs of 5' flanking sequence. Site-directed mutagenesis of the proximal promoter revealed a series of GC-rich sequences, 5 of which contributed modestly (25%) to basal hNPR-B promoter activity. Mutation of a sixth GC-rich sequence led to a >90% reduction in promoter activity. This sequence was shown to associate with Sp1 and Sp3 in vitro. The same mutation that resulted in loss of functional activity also resulted in loss of binding activity in vitro. Overexpression of Sp1 or Sp3 in Drosophila Schneider cells resulted in an increase in hNPR-B promoter activity that was completely nullified with the Sp1 binding site mutation described above. These studies provide the first description and characterization of the NPR-B gene promoter and suggest that this promoter’s activity is dominated by a single cluster of Sp1-binding elements in the proximal 5' flanking sequence of the gene.