We report here our experience in a larger series of differentiated thyroid cancer (DTC) patients who had been treated by 99m Tc-sestamibi radio-guided surgery (RGS) for 131 Iodine ( 131 I)-negative loco-regional recurrent disease.Fifty-eight patients with loco-regional 131 I-negative recurrent disease from DTC were studied with 99m Tc-sestamibi directed RGS using a hand-held 11-mm gamma probe as an intra-operative detector. Patients were selected for RGS on the basis of (a) progressive increase of serum thyroglobulin (Tg) levels after first treatment during follow-up, (b) negative high dose (100mCi, 3.7GBq) 131 I whole-body scan, and (c) positive pre-operative 99m Tc-sestamibi scintigraphy for the presence of loco-regional recurrent disease. There were 41 papillary (1 “tall” cell variant), 13 follicular and 4 Hürthle cells tumours. In 14 patients thyroid cancer recurred in the thyroid bed while cervical lymph node metastases were found in 37 patients, and 7 patients had recurrent disease both in the thyroid bed and in cervical lymph nodes.At bilateral neck exploration, 147 metastastic foci ranging from 4mm to 51mm in largest diameter (mean tumour diameter=17.3±9.5mm) were removed. Eighty-five of them (58%) had been pre-operatively identified at 99m Tc-sestamibi scintigraphy. After RGS, serum Tg levels normalised in 43 of 58 patients (serum Tg<2ng/ml – they were considered disease-free), serum Tg remained slightly increased in 12 patients without evidence of metastatic disease at scintigraphic and radiologic imaging (serum Tg<10ng/mg – they were considered living with microscopic disease), while serum Tg significantly increased up to values>900ng/ml in 3 patients who developed lung metastases. The mean lesion to background 99m Tc-sestamibi uptake ratios decreased in all 58 patients (p<0.0001). Post-surgical follow-up ranged 6–72 months (mean±SD=29.6±13.5 months). The operating surgeon assessed RGS as very useful in 14 patients in whom metastatic foci were embedded in fibrotic tissues or located behind blood vessels, useful in 22 patients, moderately useful 17 patients and not useful in 5 patients.Our data suggest that a 99m Tc-sestamibi intra-operative gamma probe can be used to identify and guide resection of recurrent loco-regional tumour in DTC patients with 131 I-negative loco-regional metastatic foci.