Localization of hyperfunctioning parathyroid glands is difficult. We examined 16 patients with biochemically proven primary or secondary hyperparathyroidism, using a 5-MHz 90 degrees real-time sector transducer. In those 16 patients, ultrasonographically guided fine-needle aspiration biopsy of 26 hypoechogenic retrothyroid nodules was performed. Hyperplastic parathyroid tissue was obtained from 24 retrothyroid nodules in 14 patients. In the other two patients, normal thyroid tissue was obtained. Both specificity and sensitivity of the aspiration biopsy technique were 100%. No complications occurred. We concluded that this technique is safe, simple, and very helpful in the localization of parathyroid tumors before surgery or before alcoholization.