The indications for stereotactic biopsies or implantation of probes for local chemotherapy in diffuse brainstem tumors have recently come under debate. The quality of performing these procedures significantly depends on the precision of the probes' placement in the brainstem. The authors evaluated the precision of brainstem probe positioning using a navigated frameless stereotactic system in an experimental setting. Using the VarioGuide stereotactic system, 33 probes were placed into a specially designed model filled with agarose. In a second experimental series, 8 anatomical specimens were implanted with a total of 32 catheters into the pontine brainstem using either a suboccipital or a precoronal entry point. Before intervention in both experimental settings, a thin-sliced CT scan for planning was obtained and fused to volumetric T1-weighted MR imaging data. After the probe positioning procedures, another CT scan and an MR image were obtained to compare the course of the catheters versus the planned trajectory. The deviation between the planned and the actual locations was measured to evaluate the precision of the navigated intervention. Using the VarioGuide system, mean total target deviations of 2.8 +/- 1.2 mm on CT scanning and 3.1 +/- 1.2 mm on MR imaging were detected with a mean catheter length of 151 +/- 6.1 mm in the agarose model. The catheter placement in the anatomical specimens revealed mean total deviations of 1.95 +/- 0.6 mm on CT scanning and 1.8 +/- 0.7 mm on MR imaging for the suboccipital approach and a mean catheter length of 59.5 +/- 4.1 mm. For the precoronal approach, deviations of 2.2 +/- 1.2 mm on CT scanning and 2.1 +/- 1.1 mm on MR imaging were measured (mean catheter length 85.9 +/- 4.7 mm). The system-based deviation of frameless stereotaxy using the VarioGuide system reveals good probe placement in deep-seated locations such as the brainstem. Therefore, the authors believe that the system can be accurately used to conduct biopsies and place probes in patients with brainstem lesions.