A spatial mismatch of up to 14 mm between optimal transcranial magnetic stimulation (TMS) site and functional magnetic resonance imaging (fMRI) signal has consistently been reported for the primary motor cortex. The underlying cause might be the effect of magnetic susceptibility around large draining veins in Gradient-Echo blood oxygenation level-dependent (GRE-BOLD) fMRI. We tested whether alternative fMRI sequences such as Spin-Echo (SE-BOLD) or Arterial Spin-Labeling (ASL) assessing cerebral blood flow (ASL-CBF) may localize neural activity closer to optimal TMS positions and primary motor cortex than GRE-BOLD. GRE-BOLD, SE-BOLD, and ASL-CBF signal changes during right thumb abductions were obtained from 15 healthy subjects at 3 Tesla. In 12 subjects, tissue at fMRI maxima was stimulated with neuronavigated TMS to compare motor-evoked potentials (MEPs). Euclidean distances between the fMRI center-of-gravity (CoG) and the TMS motor mapping CoG were calculated. Highest SE-BOLD and ASL-CBF signal changes were located in the anterior wall of the central sulcus [Brodmann Area 4 (BA4)], whereas highest GRE-BOLD signal changes were significantly closer to the gyral surface. TMS at GRE-BOLD maxima resulted in higher MEPs which might be attributed to significantly higher electric field strengths. TMS-CoGs were significantly anterior to fMRI-CoGs but distances were not statistically different across sequences. Our findings imply that spatial differences between fMRI and TMS are unlikely to be caused by spatial unspecificity of GRE-BOLD fMRI but might be attributed to other factors, e.g., interactions between TMS-induced electric field and neural tissue. Differences between techniques should be kept in mind when using fMRI coordinates as TMS (intervention) targets. Hum Brain Mapp, 2010. (c) 2010 Wiley-Liss, Inc.