The objective of this study was to determine the patient-specific optimal time to surgery preoperatively to improve operative success in gamma probe-guided parathyroid localization. Fifteen patients with hyperparathyroidism underwent a double-phase Tc99m-MIBI (15-20 mCi) parathyroid scintigraphy to study the Tc99m-MIBI clearance kinetics from parathyroid and thyroid glands and to determine the time point at which the optimal target-to-background ratio was achieved. Optimal time to surgery was determined on the basis of the time at which the target-to-background ratio was maximal. On the day of surgery the patients received the same dose of Tc99m-MIBI and were taken to the operating room at the time of optimal target-to-background ratio. A four-gland exploration was performed with identification and excision of hot lesions using a gamma probe. Ex vivo lesion-to-background ratios were determined. Histopathologic confirmation of a successful parathyroidectomy was obtained intraoperatively by frozen section. The total operative time and the time to localize and excise the parathyroid glands were recorded. The mean surgery time was compared with that of previous parathyroidectomies performed by the same surgical team without use of preoperative scintigraphy and intraoperative gamma probe. Scintigraphy correctly localized parathyroid pathology in 12 of 15 (80%) patients. Intraoperative gamma probe was successful in 15 of 15 (100%). The optimal time to surgery was found to be different for each patient, and the mean time was calculated as 136 43 minutes. Mean surgery time for the excision of parathyroid gland was 91 46 minutes, which was significantly shorter than previous four-gland exploration without use of scintigraphy and gamma probe (160 61 minutes; t = 3.021, P = 0.007). The mean ex vivo lesion-to-background gamma probe count ratio of abnormal parathyroid glands (77.35) was found to be significantly higher than that of all other lesions with abnormal Tc99m-MIBI uptake (5.05) (U = 10.5, P < 0.0001). Optimal target-to-background ratio is essential for the success of gamma probe-guided parathyroidectomy. This ratio was highly variable among the patients in this study. Individualization of the best surgical exploration time after Tc99m-MIBI injection will improve the surgical success of this operation.