Uterine leiomyomas were shown to be clonal lesions, but the relationship among different tumor nodules in multiple uterine leiomyomas remains unresolved. In this study, X-chromosomal inactivation patterns of these tumor nodules were shown by allelic polymorphism analysis through polymerase-chain reaction at the phosphoglycerate kinase and androgen receptor loci following pretreatment with the methylation-sensitive restriction enzyme HpaII or HhaI. A total number of 113 cases of uterine leiomyomas were examined. Monoclonality was demonstrated in all of the 315 nodules from 76 informative cases. The inter-nodular relationship was evaluated in 55 multiple cases with 294 tumor nodules. Different inactivation patterns were observed in 20 cases, demonstrating a multicentric origin, while an identical inactivated allele was found in all or most of the nodules in the rest of the cases, indicating a common clonal origin. The occurrence of the unicentric cases appeared to be associated with an elevated mitotic activity. Seven nodules from a multinodular case with a morphology indicative of mitotically active leiomyoma were shown to carry the identical inactivated allele, which demonstrates their unicellular origin and malignant nature. In addition, the same androgen receptor gene alteration was identified in two discrete leiomyoma nodules from a uterus. These results approve the monoclonality of uterine leiomyomas and demonstrate the presence of unicentric multiple leiomyomas.