Fine-needle aspiration (FNA) is a highly sensitive method in the differential diagnosis of thyroid nodules. However, 10% of thyroid FNAs are indeterminate for cancer, and thus additional markers may be useful diagnostically. The authors have demonstrated previously that human telomerase reverse transcriptase (hTERT) gene expression is useful in the distinction of benign lesions from malignant lesions. They therefore wondered whether the detection of hTERT gene expression was feasible using archival slides. To establish an experimental system, ribonucleic acid was extracted from human anaplastic thyroid carcinoma cell line (ARO) in cytologic specimens, and reverse transcription-polymerase chain reaction (RT-PCR) for hTERT expression was performed. RT-PCR analysis for hTERT gene detection was then performed using 58 Diff-Quik-stained archival FNA samples collected retrospectively. RT-PCR for human thyroglobulin (hTg) or beta-actin gene expression served as a positive control. Successful PCR results were obtained from 48 of the 58 cases. All 10 slides in which no RT-PCR products were noted were older than 3 years. hTERT gene expression was demonstrated in FNAs from two of seven cases (29%) of hyperplastic nodule, one of one case (100%) of Hashimoto's thyroiditis, three of eight cases (38%) of follicular adenoma, three of eight cases (38%) of Hürthle cell adenoma, three of four cases (75%) of follicular carcinoma, two of two cases (100%) of Hürthle cell carcinoma, and 11 of 18 cases (61%) of papillary carcinoma. All but one of the available 33 corresponding frozen samples exhibited the same RT-PCR results. This study demonstrates that Diff-Quik-stained thyroid FNA specimens less than 3 years old can be used for the detection of hTERT gene expression by RT-PCR. This test, along with careful cytopathologic examination, may improve our ability to differentiate benign lesions from malignant lesions in indeterminate FNA samples from thyroid nodules.