Epigenetic mechanisms of gene silencing, including promoter hypermethylation of tumor suppressor genes, have been shown to contribute to tumorigenesis. PTEN is an important tumor suppressor implicated in the pathogenesis of a number of familial and sporadic cancers. Germline mutations of PTEN predispose to dominantly inherited hamartomatous disorders Cowden syndrome and Bannayan-Zonana syndrome. Somatic PTEN mutations commonly occur in endometrial, breast, prostate, and thyroid tumors. Several investigators have speculated on PTEN promoter hypermethylation as a possible mechanism of PTEN inactivation but data supporting such observations is not forthcoming. The genomic sequence of PTEN is 98% identical to a highly conserved processed PTEN pseudogene (psiPTEN) and this sequence identity extends 841 base pairs into the promoter region. This high degree of homology has made analysis of the methylation status of the PTEN promoter quite challenging. We have investigated the methylation profiles of the promoter region of PTEN in endometrial, breast, and colon cancer cell lines, as well as in a panel of primary endometrial tumors using a combination of methylation-specific polymerase chain reaction, methylation-sensitive restriction analysis, and bisulfite sequencing. Our results show that the pseudogene, and not PTEN, is predominantly methylated in cell lines and tumors. Without careful consideration of the critical nucleotide differences between the two sequences, results obtained from PTEN analysis may not necessarily represent the methylation status of PTEN.