This study aims to evaluate the improvements of cardinal motor symptoms depending on the stimulation site relative to a standardized, reconstructed three-dimensional MRI-defined subthalamic nucleus (STN.) This retrospective, clinical study includes 22 patients with idiopathic Parkinson's disease, who consecutively underwent bilateral subthalamic nucleus stimulation. Intraoperative microelectrode recording and clinical testing were performed. The location of the best stimulation site, found intraoperatively, and the positions of the active electrode contacts 12 months after the operation were correlated to a standardized, reconstructed three-dimensional MRI-defined STN. Further, the impact of the stimulation site on rigidity, tremor and akinesia was analysed. Significant improvement of the contralateral akinesia was observed if the intraoperative stimulation site was located more lateral and superior in the MRI-STN. Furthermore, active electrode contacts located superior to or in the superior part of the MRI-STN had a significantly better effect on the tremor of the contralateral hand than in other locations inside the STN. For rigidity and akinesia, these correlations were statistically not significant. Although we found significantly better results for tremor suppression in superior and lateral aspects of the STN, for overall clinical improvement, several patients fared better with randomly distributed stimulation sites in medial, posterior or inferior parts of the MRI-defined STN. Locations of stimulation sites with the best improvements of motor symptoms were distributed randomly throughout the whole MRI-defined STN, indicating that MRI-based targeting alone is not sufficient, but intraoperative clinical testing is necessary to determine the optimal stimulation site for each individual patient.