Purpose: We investigated the biokinetics of Tc-99m-sestamibi in the thyroid of euthyroid volunteers (EVs) and in patients with autoimmune thyroid diseases and determined the best time interval between Tc-99m-sestamibi injection and calculation of uptake. Methods: Forty EVs, 30 patients with Graves' disease (GD), 15 patients with atrophic Hashimoto's thyroiditis (AHT) and 15 patients with hypertrophic Hashimoto's thyroiditis (HHT) underwent Tc-99m-sestamibi thyroid scintigraphy. Dynamic images were acquired for 20 min, and static images were obtained 20 min, 60 min and 120 min post injection. Five-, 20-, 60- and 120-min uptake, time to maximal uptake (T-max) and T-1/2 of tracer clearance were calculated. Thyroid hormones and antibodies were measured. Tc-99m-pertechnetate uptake was investigated in GD patients. Results: T-max was approximately 5 min in all four groups. The mean T-1/2 value for EVs was similar to the GD value and lower than the HHT and AHT values. The mean (+/- SD) 5-min uptake was 0.13% (+/- 0.05%) for EVs. The 5-min uptake in GD was higher than that in EVs(P < 0.001) and correlated with free thyroxine (r=0.54) and with Tc-99m-pertechnetate uptake (r=0.68). Uptake in HHT was higher than that in AHT (P=0.0003) and EVs (P=0.002). Uptake in AHT was lower than uptake in EVs (P=0.0001). Conclusion: Five minutes is the optimal time interval between Tc-99m-sestamibi injection and calculation of thyroid uptake. Five-minute uptake differentiates euthyroid individuals from GD patients. There is a high correlation between Tc-99m-sestamibi and Tc-99m-pertechnetate uptake in GD. The reduced Tc-99m-sestamibi uptake in AHT patients is probably due to glandular destruction and fibrosis. Inflammatory infiltrate and high mitochondrial density in thyrocytes possibly explain the increased uptake in GD and HHT.326702707