Hypothesis: The kinetics of technetium Tc 99m sestamibi (MIBI) in primary hyperparathyroidism are variable and affected by the cellular size of the abnormal glands, the parathyroid hormone levels, and the functional expression of P-glycoprotein (Pgp). The success of gamma probe-guided parathyroidectomy is closely related to the parathyroid-to-thyroid activity ratio at the time of surgery. Preoperative determination of maximum uptake ratio may improve the surgical outcome. Design: Thirty-one patients with primary hyperparathyroidism attributed to a solitary parathyroid adenoma (27 patients) or multiglandular hyperplasia (4 patients) underwent dynamic MIBI imaging preoperatively. Maximum MIBI activity and activity elimination half-life in the abnormal parathyroid glands and thyroid glands were measured, and the maximum uptake ratio was calculated. After a second MIBI injection on the day of surgery, all patients underwent gamma probe-guided parathyroidectomy and cervical exploration. Timing of surgery after MIBI injection was individualized according to the optimal time to surgery (time to maximum uptake ratio), which was determined by preoperative scintigraphy. During surgery, the gamma probe was used to measure ex vivo counts of excised lesions and adjacent postexcision normal tissue (background). Image characteristics, MIBI kinetics, and gamma probe findings were correlated with gland volume, oxyphil cell content, Pgp expression, and serum parathyroid hormone levels. Results: Probe localization of abnormal glands at maximum uptake ratio was successful in all patients. The volume of the parathyroid lesion ranged from 0.03 to 9.8 mL (median, 0.7 mL). Parathyroid maximum MIBI activity correlated with the volume of the gland (r = 0.54, P = .002) and serum parathyroid hormone level (r = 0.58, P = .001). No correlation between maximum MIBI activity and oxyphil cell content or Pgp expression could be demonstrated. Elimination half-life of MIBI from parathyroid inversely correlated with Pgp (r = -0.36, P = .05). The ex vivo lesion-background count ratio positively correlated with volume of the gland (r = 0.66, P = .001) and parathyroid hormone level (r = 0.48, P = .006). Ex vivo lesion counts and Pgp expression were negatively correlated (r = -0.37, P = .04). Conclusions: A strong relationship between volume of the parathyroid gland, serum parathyroid hormone levels, and MIBI uptake exists in primary hyperparathyroidism. Gamma probe-guided localization of abnormal gland(s) can be more successful if surgery is undertaken at maximum uptake ratio. High Pgp expression increases MIBI parathyroid clearance rate, decreases gamma probe counts, and may significantly alter the optimal time to surgery.