In malignant melanoma, aberrant promoter hypermethylation of tumor suppressor genes (TSG) has a crucial pathogenetic role. The aim of our study was to investigate intra-tumoral coexistence and heterogeneity of hypermethylation of different TSG promoters. We analyzed the intra-tumoral distribution of promoter methylation of p16, Rb, DAPK, RASSF1A, and MGMT in 339 assays of 33 tumors (15 melanoma primaries, 19 metastases) by methylation specific PCR. The precise function of RASSF1A as TSG involved in cell cycle regulation is not clearly understood. Therefore, we determined the phosphorylation state as a possible regulatory element of RASSF1A in a cell cycle dependent manner. We detected promoter hypermethylation of at least one gene in 76% of tumors (58%, 40%, 33%, 30%, and 20% for p16, Rb, DAPK, RASSF1A, and MGMT, respectively). 65% of cases exhibited an inhomogenous methylation pattern (48% for p16, 40% for Rb, 33% for DAPK, 20% for RASSF1A and 13% for MGMT). Samples from the core of tumors represented the methylation state of the whole tumors more accurately than samples from the edges. Local intra-tumoral correlation between methylation state of p16 and Rb and of p16 and DAPK was found. Histological results confirmed that methlytion of RASSF1A led to an aberrant expression pattern in distinct sections. Phosphorylation assays with crude extract of IGR1 cells showed that RASSF1A was phosphorylated. Intra-tumoral inhomogeneity of promoter methylation is frequent in melanoma. Heterogenous patterns suggest that promoter methylation of TSG occur late in tumorigenesis and support the hypothesis of clonal instability during progression of melanoma. The phosphorylation of RASSF1A suggests that the activity of RASSF1A is important for cell cycle progression.