The purpose of this study was to determine ability of cyst aspiration and core biopsy to differentiate malignancies, and benign lesions needing intervention from benign complex cysts in the group of Bosniak 2F and 3 renal cysts. One hundred ninety-nine indeterminate complex renal cysts were biopsied under CT or US guidance using a coaxial system (19-G sheath, 20- or 21-G Chiba or Franseen or 18-G spring biopsy needle). Cytology, amylase, lipase, LDH, fat, protein, urea nitrogen, creatine, and culture and sensitivity were assessed in all patients, histopathology in 116, and flow cytometry in 32. In 179 patients (87.9%) a definitive diagnosis was made; in 20 the specimen was inadequate. Twenty of 28 malignancies were correctly diagnosed (sensitivity 0.71); 27 of 31 benign lesions needing intervention (sensitivity 0.87) and 128 of 140 benign complex cysts (sensitivity 0.91). All inconclusive biopsies were explored revealing six malignancies. There were four CT biopsy misdiagnoses: two renal cell carcinomas; one angiomyolipoma; and one abscess. Guided cyst puncture aspiration and core biopsy significantly altered management of Bosniak 3 and 2F renal cysts obviating surgery or invasive procedures in 70% of the patients with an affirmed diagnosis of benign complex cyst and rendering timely surgical and other interventions in the remaining patients. Long-term follow-up (mean 5.6 years) revealed no further misdiagnosis.