OBJECTIVE To determine the usefulness of reverse transcriptase-polymerase chain reaction (RT-PCR) detection of parathormone (PTH) gene mRNA in needle aspirates to confirm the parathyroid nature of suspicious cervical lesions in patients with hyperparathyroidism. METHODS Ultrasound-guided fine needle aspiration cytology (FNAC) was performed on 12 patients with suspected parathyroid adenomas. The aspirates were subjected to cytologic and chromogranin A examination. Messenger RNA was isolated from left-over cells within the needle, and RT-PCR was used to amplify mRNA encoding PTH. RESULTS The 12 aspirates were positive by PTH RT-PCR, and molecular diagnosis was confirmed by histologic examination. The lower sensitivity of cytologic and immunocytochemical methods was demonstrated, respectively, by 67% and 50% of positive results. Sensitivity assays demonstrated that with our system, PTH RT-PCR products are obtained from as few as 10 pg of parathyroid mRNA. CONCLUSIONS The present study showed that RT-PCR-based analysis of PTH gene transcripts in aspirates, obtained by US-guided FNAC of cervical lesions suspicious for parathyroid adenoma, is a feasible, sensitive and specific method for preoperative diagnosis of parathyroid adenomas.