Recent findings have shown the inactivation of a Ras effector homologue gene referred to as the Ras association domain family 1 (RASSF1) gene, which is a potential human tumor suppressor gene located on chromosome 3p21.3. Hypermethylation of the CpG island promoter region of a major alternative transcript of this gene, RASSF1A, has been suggested to play a key role in pathogenesis of various carcinomas. There is limited analysis of inactivation of RASSF1A in tumors other than carcinomas. Hypermethylation of two regions of the RASSF1A CpG island was investigated in metastatic cutaneous melanomas using methylation-specific PCR; region 1 is located upstream, and region 2 is located within the first exon (1alpha) of the open reading frame of the RASSF1A transcript. Eleven melanoma cell lines and 44 melanoma tumors were examined. Methylation of RASSF1A CpG island promoter region 1 was detected in 7 (64%) cell lines and 18 (41%) tumors, and methylation of region 2 was detected in 9 (82%) cell lines and 22 (50%) tumors. Overall, RASSF1A gene hypermethylation was detected in 55% of the melanoma tumors. No methylation was detected in normal skin tissues or healthy donor lymphocytes. All cell lines that showed methylation at promoter region 1 were also methylated at promoter region 2. Hypermethylation of both CpG island regions correlated with no expression of the RASSF1A gene. RASSF1A transcripts could be reexpressed in cell lines after treatment with 5'-aza-2'-deoxycytidine. Our findings indicate that the RASSF1A gene is turned off in a significant number of melanomas and that CpG promoter region hypermethylation may play a role in the transcriptional inactivation of the RASSF1A gene in malignant melanoma.