The combination of transcranial magnetic stimulation (TMS) with functional neuroimaging has expanded the potential of TMS for human brain mapping. The precise and reliable positioning of the TMS coil is not a simple task, however. Modern frameless stereotaxic systems allow investigators to base navigation either on the subject's structural magnetic resonance imaging (MRI), functional MRI data, or the use of functional neuroimaging data from the literature, so-called “probabilistic approach.” The latter assumes consistency across individuals in the location of task-related “activations” in standardized stereotaxic space. Conventional nonstereotaxic localization of brain areas is also a common method for defining the coil position. Our aim was to evaluate the accuracy of five different localization strategies in one single study. The left primary motor cortex (left M1-Hand) was used as target region. Three approaches were based on real-time frameless stereotaxy using information based on either anatomical or functional MRI. The remaining two strategies relied either on standard cranial landmarks (i.e., the International 10–20 EEG system) or a standardized function-guided procedure (i.e., the spatial relationship between the left and right M1-Hand). The results were compared to a TMS-based mapping of the primary motor cortex; center of gravity of motor-evoked potentials (MEP-CoG) was calculated for each subject (n = 10). Our findings suggest that highest precision can be achieved with fMRI-guided stimulation, which was accurate within the range of millimeters. Very consistent results were also obtained with the “probabilistic” approach. In view of these findings, we discuss the methods and special characteristics of each localization strategy. Hum Brain Mapp, 2008. © 2007 Wiley-Liss, Inc.