A prerequisite for optimum minimally invasive radio-guided surgery (MIRS) for primary hyperparathyroidism (PHPT) is the demonstration of significant uptake of 99mTc-sestamibi in a parathyroid adenoma (PA). The aim of this study was to evaluate the clinical role or 99mTc-sestamibi SPECT in selecting patients for this procedure. Fifty-four consecutive PHPT patients were evaluated by single-session 99mTc-pertechnetate/99mTc-sestamibi planar subtraction scintigraphy, followed by 99mTc-sestamibi SPECT acquisition to localise hyperfunctioning PAs and assist in planning the surgical approach. Scintigraphy showed the presence of a solitary PA in 47/54 patients (87%) and two or more PAs in four patients (7.4%); it was negative in the remaining three patients (5.6%). The overall sensitivity of 99mTc-sestamibi scintigraphy was 94.6%. In 7/54 patients, the PA was located deep in the para-oesophageal/paratracheal space. So far, 22 patients with scintigraphic evidence of a solitary PA (in four of whom the PA was located deep in the neck) have undergone successful MIRS using the low 37 MBq (1 mCi) 99mTc-sestamibi dose protocol. Intraoperative quick parathyroid hormone (QPTH) assay demonstrated a fall in all 22 patients, thus confirming successful removal of the hyperfunctioning PA. No major surgical complications were observed. After a period of follow-up ranging between 6 and 27 months (median 13 months), no case of persistent/recurrent PHPT was recorded. When comparing the parathyroid to background (P/B) ratio measured at planar and SPECT preoperative scintigraphy with that measured intraoperatively with the gamma probe, a good linear correlation was found between the SPECT and the intraoperative gamma probe measurements (r=0.89; p<0.01) but no correlation was found with planar scintigraphic data. Our preliminary data suggest that measurement of the P/B ratio by means of 99mTc-sestamibi SPECT is more accurate in predicting the intraoperative measurements with the gamma probe. In this respect, a preoperative 99mTc-sestamibi SPECT acquisition should be recommended for better selection of PHPT patients in whom a MIRS approach can be offered.