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Ultrasonography-guided needle biopsy techniques have limitations and conventional excisional biopsy may pose challenges in the diagnosis of ultrasonography-demonstrable axillary lymphadenopathy. In this study, we report an alternative technique, radio-guided lymph node biopsy, and describe its technical aspects and diagnostic role. Between January 2006 and December 2009, six patients were included in this study (five women and one man, aged 23–65 years). After a complete clinical evaluation, all the patients were referred to our general surgery clinic for excisional lymph node biopsy from the axilla. The indications of biopsy were either the new onset of an axillary lympadenopathy on post-therapeutic screening or the presence of a persistent axillary lymphadenopathy. In all of the cases, ultrasonography confirmed the clinical suspicion of axillary lymphadenopathy and the radio-guided lymph node biopsy technique was used for the diagnosis. Tc-99m-labelled human serum albumin macroagregate in saline was injected under ultrasonographic guidance for the localization of the lymph nodes. A γ-probe was used to guide the excisions.The lymph nodes that were involved were successfully localized and excised surgically. The postoperative course was uneventful and no complications occurred in all cases. Pathological examination of the excised lymph nodes showed reactive hyperplasia in three patients, tuberculous lymphadenitis in one patient, Hodgkin's lymphoma in another and, non-Hodgkin's lymphoma in one patient. Radio-guided lymph node biopsy has proved to be an accurate and a safe technique for the diagnosis of axillary lymphadenopathies in the indicated subset of patients.