Background. Primary hyperparathyroidism is the leading cause of hypercalcemia in the United States. The goal of this study was to evaluate the feasibility of radioguided thoracoscopic mediastinal parathyroidectomy and intraoperative immunoreactive parathyroid hormone (iPTH) level testing to guide completeness of resection. Methods. Mediastinal parathyroidectomy was performed thoracoscopically with intraoperative radioguidance using a hand-held gamma probe after injection of 10 mci of TC-99m sestamibi. Parathyroid excision was confirmed by ex vivo measurement of specimen radioactivity greater than 20% of background. Complete resection was confirmed by a greater than 50% decrease in serum iPTH level at 5 minutes postresection. Results. Four patients had mediastinal parathyroid glands successfully localized and resected thoracoscopically. Mean weight of the excised parathyroid adenoma was 1,714 mg (range, 425 to 4,400 mg). Baseline iPTH levels decreased from a mean of 202 to 39 pg/dL 5 minutes postresection. One patient underwent radioguided resection of a second enlarged cervical parathyroid adenoma at the same setting when his intraoperative iPTH levels failed to fall below 50% of baseline, despite resection of a 440 mg mediastinal parathyroid gland. Median hospital stay was one day. All mediastinal parathyroid glands resected were confirmed adenomas on final histologic examination. All patients were normocalcemic at follow-up (mean, 25 months), indicating cure. Conclusions. Thoracoscopic mediastinal parathyroidectomy with intraoperative iPTH level monitoring is safe and effective. Radioguidance facilitates parathyroid localization. Ex vivo specimen radioactivity of greater than 20% of background confirms parathyroid resection and obviates the need for costly, time-consuming frozen section analysis. A 50% decrease in baseline iPTH level 5 minutes postresection confirms complete resection of parathyroid adenomas. © 2005 by The Society of Thoracic Surgeons.