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Proposed mechanisms of parathyroid localization in ‘dual-phase’ technetium-99m-sestamibi imaging include increased presence of mitochondria leading to greater uptake and slower washout compared with thyroid tissues owing to reduced expression of P-glycoprotein. Using new techniques of quantitative single-photon emission computed tomography (SPECT)/computed tomography (CT), we have measured MIBI uptake and washout to better understand factors related to conspicuity.We retrospectively reviewed 125 consecutive patients. Early and delayed SPECT/CT images were reconstructed using a previously validated technique. Maximum standardized uptake values of parathyroid adenomas and thyroid tissue were measured, and corresponding washout rates were calculated.Of 53 patients with localization of parathyroid adenoma (42%), median maximum standardized uptake values were higher for parathyroid adenomas than for thyroid tissue on both early (6.43±3.78 vs. 4.43±1.93, P<0.001) and delayed (3.40±3.09 vs. 1.84±1.05, P<0.001) images, being true on a per-patient basis in 41 (77%) and 48 (91%) patients, respectively. Median washout rates were slower from parathyroid adenomas than from thyroid lobes (0.26±0.16 vs. 0.42±0.18 h−1, P<0.001), being true on a per-patient basis in 43 (81%) patients. Similar findings were observed in a subgroup of 37 patients with surgically-confirmed adenomas. Of the patients where initial parathyroid uptake did not exceed thyroid uptake, all 12 exhibited slower MIBI washout from the parathyroid adenomas than from the thyroid gland. The characteristics of the thyroid gland did not differ in 67 patients without parathyroid localization.Quantitative analysis of 53 patients with localization of parathyroid adenoma revealed both a generally higher initial absolute uptake and slower rate of washout of MIBI in parathyroid adenomas than in thyroid tissue. The findings may support the hypothesis that both mechanisms proposed for parathyroid conspicuity in the dual-phase examination increased mitochondrial binding and slower washout owing to reduced P-glycoprotein expression. The technique of quantitative SPECT/CT represents a powerful tool for measuring tissue uptake to elucidate the contribution of these factors to lesion conspicuity.