Aim of the present study was to investigate the feasibility and long-term results of focused radioguided parathyroidectomy using a "low" 37 MBq (1 mCi) 99mTc-sestamibi dose protocol compared to conventional "high 740 MBq (20 mCi) 99mTc-sestamibi dose protocol" in patients with primary hyperparathyroidism (PHPT). The data of focused radioguided surgery obtained in a group of 320 consecutive PHPT patients with high probability of the presence of a solitary parathyroid adenoma (PA) were studied. All patients underwent preoperative imaging work-up of double-tracer 99mTc-pertechnetate/99mTc-sestamibi subtraction parathyroid scintigraphy (Sestamibi scintigraphy) and high resolution neck ultrasound (US). In 301/320 patients (96.6%) focused minimally invasive radioguided surgery was successfully performed by administering a "low" 37 MBq (1 mCi) 99mTc-sestamibi dose in the operating room 10 minutes before operation. No major intraoperative complications were recorded. Focused radioguided surgery required a mean time of 32 min and a mean hospital stay of 1.2 days. Local anesthesia was applied in 75 patients, 66 of whom (88%) were patients older than 65 years with comorbidities contraindicating general anesthesia. No case of persistent or recurrent PHPT was observed during post-surgical follow-up (range = 18-70 months; mean +/- SD = 15.3 +/- 9.1 months). Radiation exposure dose to the operating surgeon was 1.2 microSi/hour with the "low 37 MBq (1 mCi) 99mTc-sestamibi dose", and less than 1.0 microSi/hour for the other operating-room personnel. Focused low dose radioguided parathyroidectomy is a safe and effective means to localize parathyroid adenomas in patients affected by solitary PA thus reducing by 20 fold the radiation exposure dose to the patients and operating room personnel.