Germline mutations in both alleles of the thyroid peroxidase (TPO) gene have been reported as a frequent cause of congenital hypothyroidism resulting from a total iodide organification defect (TIOD). Because TPO mutations have a prevalence of 1 in 66,000 newborns and is inherited in an autosomal recessive mode the frequency of a heterozygous germline mutation in the TPO gene should reach about 1 in 260 in the population. A somatic TPO mutation coinciding with a somatic loss of one of the TPO alleles or a TPO germline mutation could lead to somatic loss of TPO activity with impairment of thyroid hormone synthesis and decrease of growth control. The latter would lead to increased thyroid epithelial cell proliferation and the subsequent development of a scintigraphically cold thyroid nodule (CTN). To test this hypothesis we studied 40 CTN for the presence of mutations or loss of heterozygosity (LOH) in the TPO gene. For comparisons we also studied LOH in 17 autonomously functioning thyroid nodules (AFTN). Genomic DNA was extracted from nodular and surrounding tissue, polymerase chain reaction (PCR) amplified, sequenced, and analyzed for LOH. In 6 CTNs of 37 informative cases we detected LOH using the genomic markers sRA, D2S2268, and D2S319 within or near the TPO gene locus (2p24-25). In contrast, a genomic marker closer to the centromer (D2S144, 2p24-21) shows LOH in only 1 CTN. We did not detect LOH in AFTN. In none of the cases a germline or somatic mutation in the TPO gene was detectable in the TPO gene. LOH in 6 of 37 CTNs suggests that genetic defects at the TPO or the chromosomal locus 2p24-25 might play a role in the etiology of CTNs. However, we did not find the combination of LOH with a somatic mutation in the TPO gene. It is therefore likely that a gene defect near the TPO locus is part of the neoplastic process in a subgroup of CTNs.