Both hypermethylation of the tumor suppressor gene RASSF1A and activating mutations of the KRAS and/or BRAF gene have been reported in a variety of human cancers. To investigate these epigenetic and genetic alterations in endometrial carcinoma (EC), we examined their frequency in 4 uterine EC cell lines and in 75 sporadic primary ECs. Using methylation specific PCR, we found RASSF1A methylation in 25 of 75 (33.3%) ECs. RASSF1A methylation was significantly associated with microsatellite instability (MSI, p < 0.001) and also with hMLH1 methylation (p < 0.001). KRAS mutations were detected in 14 of 75 (18.7%) ECs. BRAF mutations were identified in only 3 of 75 (4.0%) ECs and were not found in ECs with KRAS mutations or RASSF1A methylation. RASSF1A methylation was more frequent in KRAS mutation-negative ECs than in KRAS mutation-positive ECs (37.7% vs 14.3%), but this inverse correlation is not statistically significant (p = 0.122). However, we observed that RASSF1A methylation was inversely correlated with KRAS and/or BRAF mutations (p = 0.028) in MSI-negative ECs, while this inverse correlation disappeared in MSI-positive ECs. Furthermore, in MSI-positive ECs, 2 cases of concomitant RASSF1A methylation and KRAS mutation were found. Taken together, these results provide strong evidence that, in EC tumorigenesis, RASSF1A promoter hypermethylation is as important as KRAS mutations in activating the RAS pathway.