Polymerase chain reaction (PCR) amplification has been used to determine the clonal composition of tissues based on analysis of the pattern of X-chromosome inactivation, but its use has been limited by technical difficulties. This report presents an expedited method to use PCR in the analysis of clonality. The method uses gel electrophoresis of heteroduplexes formed with an artificial heteroduplex generator (HG) and PCR products from the phosphoglycerate kinase-1 (PGK-1) gene from the tissue sections. Amplification was successful in 36 of 37 cases originally diagnosed as endometrial adenocarcinoma. HG analysis of 36 cases confirmed heterozygosity in 12 cases (33.3%). PGK-1 PCR amplification product was obtained from both control and lesional tissue in 10 of the 12 heterozygous cases. Of these 10 cases, seven were shown to consist of clonal cell populations by HG analysis. Two of three cases diagnosed as well-differentiated endometrioid adenocarcinoma were found to be comprised of polyclonal populations of cells. One case produced an anomalous pattern with HG analysis and was shown to be aneuploid by fluorescence in situ hybridization (FISH) with a chromosome X alpha-satellite probe. It is concluded that HG is a useful alternative to restriction fragment length polymorphism (RFLP) analysis of X-chromosome inactivation as a marker of tissue clonality in cases in women.