Background: Radioguided parathyroidectomy using TC-99m-sestamibi injection and the handheld gamma probe allows more precise and rapid intraoperative localization of abnormal parathyroid glands. This technique is based on the principle that hypercellular parathyroid tissues have markedly higher in vivo radiotracer counts than surrounding tissue including thyroid and lymph nodes. While in vivo radioactivity after TC-99m-sestamibi administration in various tissues has been documented, there is a lack of data regarding ex vivo radioactive properties after surgical resection. Methods: During a 6-week period in June/July 2005, 21 patients underwent radioguided parathyroidectomy by a single surgeon. Fifty-four tissue samples (39 parathyroid, 15 nonparathyroid) from these patients were collected and analyzed for ex vivo radioactive counts over a 30-min period. These data were then compared with the pathologic results. Results: There is a significant difference in ex vivo counts between parathyroid adenomas, hyperplastic glands, and nonparathyroid tissue immediately after resection. However, radioactive decay/slope rates do not differ between the tissues. Importantly, an ex vivo count of >20% of background is 100% specific for parathyroid tissue. These differences persisted for up to 30 min. Conclusions: This is the first comprehensive study of ex vivo radioactive properties after TC-99m-sestamibi injection during radioguided parathyroidectomy. Parathyroids have a greater rate of uptake compared to nonparathyroid tissue, allowing ex vivo counts to predict tissue type. These tissues have similar decay rates, allowing these predictions to be made anytime up to 30 min after gland resection. © 2006 Elsevier Inc. All rights reserved.