We determined clonality of thyroid tumors from female patients who had restriction fragment length polymorphisms (RFLP) in the X chromosome genes hypoxanthine phosphoribosyltransferase (HPRT) or phosphoglycerate kinase (PGK). We screened normal thyroid tissue from 59 female patients; of the informative cases 14 were heterozygous for a Bgl I site on PGK and 4 were heterozygous for a Bam HI site on HPRT. In monoclonal tumors, one of the polymorphic alleles was selectively digested after additional digestion with Hpa II, a methylation sensitive enzyme, whereas in polyclonal tissue both were decreased to a similar extent. Normal thyroid tissue from all patients showed a polyclonal pattern. Of the 18 tumors studied, 12 were solitary thyroid nodules, and 6 were obtained from multinodular goiters (MNG). The following were monoclonal: 6/6 follicular adenomas, 2/2 follicular carcinomas, and 1/1 anaplastic carcinoma. Two of the three papillary carcinomas showed intermediate patterns, possibly due to contaminating effects of stromal tissue present in most of these neoplasms. Of the six nodules from MNG, four were polyclonal. The two largest gave a distinct monoclonal pattern. Most solitary thyroid tumors are monoclonal, supporting a somatic cell mutation model of thyroid neoplasm formation. Nodules from MNG are largely hyperplastic, although monoclonal neoplasms do occasionally arise within these glands. The specific somatic mutations leading to clonal expansion and determination of tumor phenotype are presently unknown.