To determine whether a tumor suppressor gene of importance to epithelial ovarian cancer resides on the X chromosome, we examined loss of heterozygosity (LOH) in 123 epithelial ovarian cancer cases. In 54 such cases, we examined LOH at 26 loci on the human X chromosome. In eight cases, we examined LOH in 14 loci and in 61 cases we examined LOH in 13 loci. Matched DNA samples from tumors and corresponding normal tissues were analyzed by polymerase chain reaction (PCR) amplification of microsatellite markers. Frequent losses were found in epithelial carcinomas at the Xq25-26.l region, including DXS1206 (34.5% loss in informative cases), DXS1047 (27.7%), HPRT (24.1%), and DXS1062 (33.3%). The minimum overlapping region of LOH was approximately 5 megabases (Mb), flanked by DXS1206 (Xq25) and HPRT (Xq26.1). The methylation status of the remaining allele of the androgen receptor gene in the tumors exhibiting LOH at the Xq25-26.1 region suggested that the loss was exclusively in the inactive X chromosome. We next determined whether a significant relationship exists between Xq LOH and other parameters, including histologic grade and/or clinical stage of the tumors and LOH at TP53. The Xq LOH had a significant association with grade 2 to 3 tumors at stages II to IV. Sixteen of 18 cases that showed Xq LOH revealed LOH at the TP53 locus, and 45% of tumors exhibiting LOH at TP53 showed Xq LOH. These results suggest that there may be a tumor suppressor gene or genes which escape inactivation of the X chromosome at Xq25-26.1, and that the loss of the gene(s) at Xq25-26.1 is frequently accompanied by loss of the TP53 or loss of another gene on chromosome 17. These losses may contribute to the progression from a well-differentiated to a more poorly differentiated state or to metastatic aggressiveness.