Clinical and experimental data suggest a low thyroid hormone synthesis in cold thyroid nodules (CTN). Therefore, the Na(+)/I(-)-symporter (NIS) as the first step in the thyroid hormone synthesis could be a possible candidate gene in the pathogenesis of CTNs. A reduction of NIS transcripts in CTNs compared to samples of normal thyroid tissues with large inter-individual variations ranging from 2- to 700-fold reductions was observed with real-time RT-PCR. Therefore, the aim of our investigations was to perform an intra-individual comparison of NIS expression in CTNs. Moreover, we used direct detection of NIS mRNA by RNase protection assay (RPA). We investigated 14 patients with one CTN for NIS mRNA expression. NIS mRNA transcripts from nodule and surrounding tissue were examined by RPA. A significantly reduced NIS expression was detected in 86% of the CTNs compared to their corresponding surrounding tissue. The level of NIS expression was decreased to more than 65% in 10 CTNs (72% of the nodules). Two of the 14 nodules showed a decrease of NIS mRNA expression of 42%, and 32%, while no significant differences could be detected in 2 cold nodules. Compared to other studies the intra-individual comparison of NIS mRNA expression revealed a much lower variation of reduced NIS expression in CTNs. Further studies should try to identify molecular factors like post-transcriptional modifications or alterations in iodide organification which are likely to be involved in the pathogenesis of CTNs.