Precise needle puncture of the renal collecting system is an essential but challenging step for successful percutaneous nephrolithotomy. We evaluated the efficiency of a new real-time electromagnetic tracking system for in vivo kidney puncture. Six anesthetized female pigs underwent ureterorenoscopy to place a catheter with an electromagnetic tracking sensor into the desired puncture site and ascertain puncture success. A tracked needle with a similar electromagnetic tracking sensor was subsequently navigated into the sensor in the catheter. Four punctures were performed by each of 2 surgeons in each pig, including 1 each in the kidney, middle ureter, and right and left sides. Outcome measurements were the number of attempts and the time needed to evaluate the virtual trajectory and perform percutaneous puncture. A total of 24 punctures were easily performed without complication. Surgeons required more time to evaluate the trajectory during ureteral than kidney puncture (median 15 seconds, range 14 to 18 vs 13, range 11 to 16, p=0.1). Median renal and ureteral puncture time was 19 (range 14 to 45) and 51 seconds (range 45 to 67), respectively (p=0.003). Two attempts were needed to achieve a successful ureteral puncture. The technique requires the presence of a renal stone for testing. The proposed electromagnetic tracking solution for renal collecting system puncture proved to be highly accurate, simple and quick. This method might represent a paradigm shift in percutaneous kidney access techniques.