Background: Isolated rib lesions detected on bone scanning can pose a diagnostic challenge, particularly in patients with a known primary cancer. The purpose of our study was to assess the diagnostic value of radionuclide-guided rib biopsy with an intraoperative gamma probe. Methods: We conducted a retrospective chart review of 10 patients who underwent an intraoperative gamma probe-guided rib biopsy. Rib defects were identified by radionuclide bone scan. Patients received an intravenous dose of technetium 99m methylene diphosphonate within 6 hours before surgery. The approximate location of the rib defect was scanned with a hand-held gamma probe intraoperatively; an excisional rib biopsy was performed in the area with the highest tracer activity. Results: Median age was 54 years (range, 40 to 83) and median body mass index was 32.5 (range, 23 to 52). Seven patients had a known primary extraosseous cancer and 3 patients had no history of cancer. Seven patients had pain at the time of initial evaluation. The median operative time was 45 minutes, median operative blood loss was 25 mL, and median length of stay was 1 day. Complications occurred in 2 patients: a pneumothorax that did not require treatment and a delayed hemothorax that required tube thoracostomy. Rib biopsy was accurate in all 10 patients; metastatic disease was found in 5 of 7 cancer patients. Pain improved or resolved in 4 patients. Conclusions: The evaluation of isolated rib lesions using radionuclide-guided rib biopsy with an intraoperative hand-held gamma probe is accurate and potentially therapeutic. Thoracic surgeons and oncologists should be aware of this approach.