Background: Tc-99-sestamibi scanning is utilized to determine whether patients with primary hyperparathyroidism (HPT) are candidates for minimally invasive parathyroidectomy (MIP). However, if the result of this scan is negative, many surgeons recommend bilateral parathyroid exploration because of possible multi-gland disease. The objectives of this study were to determine whether patients with primary HPT and negative sestamibi scans can benefit from additional imaging studies and are still potential candidates for MIP. Methods: Between March 2001 and April 2006, 578 consecutive patients with HPT underwent parathyroidectomy by a single surgeon. Pre-operative sestamibi scans had been done in 458 (79%) of these patients, 90 (20%) of which had negative results. These patients formed our study cohort. Results: Of the 90 patients, 60 (67%) had a single adenoma, 17 (19%) double adenomas and 13 (14%) 3- to 4-gland hyperplasias. In 74 (82%) patients, localization was further investigated with one or more studies including thallium subtraction scans (n = 30), ultrasound (n = 15) and intra-operative internal jugular venous sampling (n = 49). Additionally, the use of radio-guided techniques intra-operatively facilitated minimally invasive techniques. Of these 90 patients, 47 had positive results from preoperative studies, including 12 positive thallium, 5 positive ultrasound and 13 positive internal jugular sampling results. In addition, positive results were observed for 17 patients using radio-probe techniques in the operating room. Accordingly, minimally invasive techniques were attempted in these 47 patients; 42 (89%) had single adenomas and in 5 the technique was converted to bilateral exploration for double adenoma/hyperplasia. In the setting of a negative sestamibi, the sensitivities of thallium scans and ultrasound were 30% and 27%, respectively. The overall cure rate in the 90 patients with negative sestamibi scans was 99%. Conclusion: In patients with primary HPT and a negative sestamibi scan, most patients (67%) will have a single adenoma. These patients benefitted from additional localization tests, which yielded a positive result in 52% of patients. Therefore, even in the setting of a negative sestamibi scan, the majority of patients with primary HPT are still candidates for MIP. © 2007 Society of Surgical Oncology.