RASSF2 is a recently identified member of a class of novel tumour suppressor genes, all containing a ras-association domain. RASSF2 resides at 20p13, a region frequently lost in human cancers. In this report we investigated methylation status of the RASSF2 promoter CpG island in a series of breast, ovarian and non-small cell lung cancers (NSCLC). RASSF2 was frequently methylated in breast tumour cell lines (65%, 13/20) and in primary breast tumours (38%, 15/40). RASSF2 expression could be switched back on in methylated breast tumour cell lines after treatment with 5'-aza-2'deoxycytidine. RASSF2 was also frequently methylated in NSCLC tumours (44%, (22/50). The small number of corresponding normal breast and lung tissue DNA samples analysed were unmethylated. We also did not detect RASSF2 methylation in ovarian tumours (0/17). Furthermore no mutations were found in the coding region of RASSF2 in these ovarian tumours. We identified a highly conserved putative bipartite nuclear localization signal (NLS) and demonstrated that endogenous RASSF2 localized to the nucleus. Mutation of the putative NLS abolished the nuclear localization. RASSF2 suppressed breast tumour cell growth in vitro and in vivo, while the ability of NLS-mutant RASSF2 to suppress growth was much diminished. Hence we demonstrate that RASSF2 has a functional NLS that is important for its tumour suppressor gene function. Our data from this and a previous report indicate that RASSF2 is frequently methylated in colorectal, breast and NSCLC tumours. We have identified RASSF2 as a novel methylation marker for multiple malignancies and it has the potential to be developed into a valuable marker for screening several cancers in parallel using promoter hypermethylation profiles.