Previous studies have shown a significant association between allelic frequencies at the ANP gene locus and aldosterone responsiveness to angiotensin in aldosterone-producing adenoma (APA). We searched for any gross insertions or deletions in the ANP gene in APA and any associations between allelic frequencies at the Hpa II and Sca I RFLP sites within the ANP gene and angiotensin-responsive and unresponsive APA and normal subjects. We also searched for possible point mutations in the promoter region of the ANP gene (-595 to transcription start site) in peripheral blood and tumor DNA from 59 patients with APA and in peripheral blood DNA from 39 normal subjects by polymerase chain reaction and single strand conformation polymorphism (PCR-SSCP) analysis. No large alterations in the ANP gene were observed, and no difference in allelic frequencies at the RFLP sites were seen between the two tumor subtypes, angiotensin-responsive and angiotensin-unresponsive APA, or between the APA group and normal subjects. SSCP analysis, however, did reveal mutations in the promoter region of the ANP gene (-375 to -595) in both peripheral blood and tumor DNA from 8 of 59 (14%) patients with APA, compared with only one of 39 normal controls (2.6%). This study suggests that alterations in the proximal promoter region of the ANP gene in APA may be important in the regulation of ANP transcription and may be involved in the underlying pathophysiology of aldosterone-producing adenoma in at least some patients.