Abstract--Previous studies have shown that atrial natriuretic peptide (ANP) can inhibit transcription of its receptor, guanylyl cyclase A, by a mechanism dependent on cGMP and have suggested the presence of a putative cGMP-response element (cGMP-RE) in the Npr1 gene promoter. To localize and characterize the putative cis-acting element, we have subcloned a 1520-bp fragment of the rat Npr1 promoter in an expression vector containing the luciferase reporter gene. Several fragments, generated by exonuclease III-directed deletions, were transiently transfected into cells to measure their promoter activity. Deletion from -1520 to -1396 of a 1520-bp-long Npr1 promoter led to a 5-fold increase in luciferase activity. Subsequent deletion to the position -1307 resulted in a decrease of luciferase activity by 90%. Preincubation of cells with 100 nM of ANP or 100 µM 8-bromo-cGMP inhibited luciferase activity of the 1520-bp and 1396-bp-long fragments, but not the activity of the 1307-bp fragment, suggesting that the cGMP-RE is localized between positions -1396 and -1307. The cGMP regulatory region was narrowed by gel shift assays and footprinting to position -1372 to -1354 from the transcription start site of Npr1 and indicated its interaction with transcriptional factor(s). Cross-competition experiments with mutated oligonucleotides led to the definition of a consensus sequence (-1372 AaAtRKaNTTCaAcAKTY -1354) for the novel cGMP-RE, which is conserved in the human (75% identity) and mouse (95% identity) Npr1 promoters.