Clonality of archival formalin-fixed tissue sections was analyzed by polymerase chain reaction amplification of a portion of the X-linked phosphoglycerate kinase (PGK-1) gene. Amplification was successful in 29 of 36 cases of uterine endometrioid adenocarcinoma. Five of these cases, including both tumor and control tissue from the same patients, were heterozygous for the BstXI polymorphic site of the PGK-1-amplified product, permitting analysis of clonality. Pretreatment of the DNA with HpaII blocked amplification of one of the two PGK-1 alleles from four of five cases of tumor, indicating the clonal pattern of X chromosome inactivation in these cases. In contrast, in DNA from paired control tissues HpaII pretreatment had no effect, indicating a random pattern of X chromosome inactivation in normal tissue. One of the cases of endometrioid adenocarcinoma contained a high proportion (45%) of nontumor cells, precluding the determination of clonality. We conclude that polymerase chain reaction amplification can be used for the determination of the pattern of X chromosome inactivation in formalin-fixed tissue sections. Such an approach makes it feasible to include specimens from archival tissue collections in the analysis of clonality.