Accumulated data using functional, morphologic, and histochemical analysis suggests that follicular proliferations in the thyroid include polyclonal and monoclonal patterns with encapsulated follicular adenomas most frequently monoclonal, and other nodules generally polyclonal. However, examples of polyclonal carcinomas or adenomas raise the possibility that histologically similar lesions may arise through different pathogenetic mechanisms. The authors have performed a clonal analysis of histologically benign and malignant thyroid nodules in seven women using HPRT (hypoxanthine phosphoribosyl transferase) and PGK (phosphoglycerate kinase) restriction fragment length polymorphisms (RFLPs) on the X chromosome. These RFLPs used in concert with methylation-sensitive restriction endonucleases HpaII and HhaI permit distinction of active and inactive X chromosomes. DNA from a multinodular goiter showed equal sensitivity of both X chromosome RFLP alleles to a methylation-sensitive restriction endonuclease, consistent with a polyclonal origin. In contrast, three solitary follicular nodules and three carcinomas displayed predominant sensitivity of a single RFLP allele, consistent with a monoclonal origin. Although further detailed studies will be necessary to understand polyclonal origins reported for some adenomas, our data from a limited number of samples supports a predominantly monoclonal origin, and possible neoplastic pathogenesis, for many solitary adenomatous nodules in the thyroid.