Purpose: Double-phase scintigraphy with Tc-99m sestamibi is a good method to detect hyperfunctioning parathyroid glands. This study tried to determine the best time for delayed images: 2 or 4 hours. Methods: Fifty-six patients were studied, and 35 of them had primary hyperparathyroidism (mean age, 53 +/- 13 years; 54% were women). Cervical ultrasonography was performed on 29 of 56 (52%) patients and surgery in 16 of 56 (29%) patients. The dose was 740 MBq (20 mCi) given intravenously, and the acquisition was performed at 10 minutes, 2 h, and 4 h using anterior views, including the mediastinum. Studies that had positive results were analyzed blindly by two independent observers, who selected the best definition for abnormal activity. Results: Nineteen of 56 (34%) studies were negative and 37 of 56 (66%) were positive, 25 of them with one focus and 12 with two or more parathyroid foci. Analysis revealed 76% agreement between the observers (the rest was classified by consensus). In 70% of the cases, the best delayed image was obtained at 2 hours, in 16% at 4 hours (P < 0.00001), and in 14% both images were similar. In those cases with better images at 4 hours, the 2-hour images also showed the lesions. In two patients, lesions were seen only at 2 hours. These results could be explained by tracer decay, washout of parathyroid activity, or both. Conclusions: The best protocol should include the early 10-minute image and the 2-hour delayed view. Further controls do not appear necessary. This may be important for patient throughput.