Hypermethylation of CpG island promoters is associated with transcriptional inactivation of tumor suppressor genes in neoplasia. Inactivation of p16 and Pten was related to the development of pheochromocytomas. In this report, we investigated the methylation status of the p16INK4a cell cycle inhibitor gene and other prominent tumor-related genes ( PTEN, RASSF1 A, CDH1, MSH2, MLH1, VHL, and TIMP3) in sporadic and multiple endocrine neoplasia type 2 (MEN2) pheochromocytomas by methylation-specific PCR. Hypermethylation was detected in 48 % of pheochromocytomas for RASSF1 A, 24 % for p16, 36 % for MSH2, 16 % for CDH1, and 8 % for PTEN. No VHL, MLH1, and TIMP3 methylation was observed. Interestingly, the frequency of p16 inactivation in familial tumors was higher (5 out of 12, 42 %) than in sporadic tumors (1 out of 13, 8 %; p = 0.047) and RASSF1 A inactivation was more common in the hereditary tumors (58 %) compared to the sporadic tumors (38 %). Combined methylation of RASSF1 A and p16 was found only in MEN2-related pheochromocytomas. Thus, a subset of hereditary pheochromocytomas displays preferential methylation of p16 and RASSF1 A.