Aberrant methylation of gene promoter regions is one of the mechanisms for inactivation of tumor suppressor genes in human malignancies. In this study, the methylation pattern of 24 tumor suppressor genes was analyzed in 75 samples of ovarian cancer using the methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay. Of the 24 tumor suppressor genes examined, aberrant methylation was observed in 17. The three most frequently methylated genes were CDKN2B, CDH13 and RASSF1, followed by ESR1 and MLH1. Methylation frequencies ranged from 1.3% for CDKN2A, RARβ, CASP8, VHL and TP73 to 24% for CDKN2B. The corresponding normal DNA from each patient was also investigated. Methylation was detected in tumors, although not in normal tissues, with the exception of two samples, indicating aberrant methylation in tumors. Clear cell carcinoma samples exhibited a higher frequency of CDKN2B promoter hypermethylation compared to those of other histological types (P=0.05). Our data indicate that methylation of the CDKN2B gene is a frequent event in ovarian carcinogenesis and that analysis of only three genes is sufficient to detect the presence of methylation in 35% of ovarian cancer cases. However, more studies using a much larger sample size are needed to define the potential role of DNA methylation as a marker for ovarian cancer.