In the present study we investigated the role of radio-guided surgery with Iodine-131 (I-131) in a group of 31 patients with differentiated thyroid cancer (DTC) and loco-regional recurrent disease. The principal inclusion criterion for I-131 radio-guided surgery in our protocol was the presence of an I-131 positive loco-regional disease relapse after previous total thyroidectomy and at least 2 ineffective conventional I-131 treatments. The protocol we used consisted of the following steps. Day 0: all patients were hospitalized and received a therapeutic 3.7 GBq (100 mCi) dose of I-131 after thyroid hormone therapy withdrawal in condition of overt hypothyroidism (serum TSH levels>30 microUI/ml). Day 3: a whole body scan following the therapeutic I-131 dose (TxWBS) administration was acquired. Day 5: neck surgery was performed through a wide bilateral neck exploration using a 15-mm collimated gamma probe, measuring the absolute intra-operative counts and calculating the lesion to background (L/B) ratio. Day 7: post-surgery TxWBS was performed using the remaining radioactivity to evaluate the completeness of tumoral lesions extirpation. The final histologic examination showed the presence of 184 metastatic foci; among them, 98 (53.2%) were evident by both TxWBS and gamma probe evaluation, 76 (41.3%) were demonstrated only by gamma probe, and 10 (5.4%) were negative by both TxWBS and gamma probe evaluation. During follow-up (8 months to 4.9 years, mean 2.8 years), DxWBS, serum Tg levels off l-T4, and US showed absence of loco-regional disease in 25 patients (80.6%) while 6 patients had persistent disease. In conclusion, this protocol allowed us to identify neoplastic foci with high sensitivity and specificity, enabling us to remove loco-regional I-131 disease recurrences resistant to previous conventional I-131 therapies. Furthermore, the gamma probe allowed detection of some additional tumoral foci in sclerotic areas or located behind vascular structures that were not visualized at the pre-surgery TxWBS evaluation.