Ultrasound-guided (US) fine-needle aspiration (FNA) can successfully localize abnormal parathyroid tissue (PT) preoperatively in hyperparathyroid patients. Samples from 12 patients with primary hyperparathyroidism evaluated using this technique since 1990 at the National Institutes of Health form the basis of this report. Eleven patients had undergone previous parathyroid surgery that failed to correct their hyperparathyroidism. Cytological evaluation and C-terminal (midmolecule) parathyroid hormone radioimmunoassay (PTH RIA) were performed on all samples. When sufficient material was available, immunocytochemical stains for chromogranin and thyroglobulin were performed. All cytological diagnoses were made with-out knowledge of the PTH RIA results. Using a combined approach of cytology and immunocytochemistry, six of 12 of the samples (50%) were diagnosed as PT. Follow-up on these six patients was confirmatory. Four of 12 samples (33%) were identified as thyroid; one of these patients had a PT adenoma identified in another location (the remaining three patients await further localization studies). Two of 12 samples (17%) could not be diagnosed because of insufficient cellularity; in both patients PT lesions were found in other locations. Morphological features of PT in FNA include the presence of cellular tissue fragments with epithelial cells arranged perivascularly around capillary cores, an overall organoid or trabecular architecture, and frequent microacini. Parathyroid tissue cells have round, fairly uniform nuclei measuring 6 to 8 microns. Clusters of larger oxyphil cells may show considerable anisonucleosis. The absence of features of thyroid tissue such as hemosiderin-laden macrophages, abundant colloid, and paravacuolar granules is significant. However, in cases of intrathyroidal PT, admixed thyroid material included in the aspiration tract may be present immunocytochemical stains for chromogranin, which is present in parathyroid tissue but not thyroid follicular cells, were positive in six of six samples interpreted as PT by cytology. No thyroglobulin staining was observed in any of the four of six PT samples for which material was available. C-terminal (midmolecule) PTH RIA correlated with cytological diagnoses in 100% of samples. Parathyroid hormone levels ranged from 1,300 to 262,000 pg/mL (normal blood level, 50 to 340 pg/mL) in the six samples diagnosed as PT by cytology. Parathyroid hormone RIA levels in the six non-PT samples were below normal blood values. The combined approach of cytology and immunocytochemistry provides high diagnostic accuracy in the interpretation of US-guided FNA for preoperative localization of parathyroid tissue.