Cortical motor representation of 12 muscles of the trunk and the upper and lower extremity was investigated in 18 healthy subjects using focal transcranial magnetic stimulation (TMS) in conjunction with a frameless stereotactic system (FSS). This combination allowed us to orientate stimulation sites to the individual central sulcus rather than to bony landmarks. Distinct but overlapping areas of muscle representation were identified and the 3-dimensional representation of those 12 muscles along the course of the central sulcus was obtained. With increasing stimulus intensity, the cortical output maps changed in that more muscles became excitable, motor evoked potential (MEP) amplitude and size of the responsive area increased and latency of the MEP decreased. These effects were more pronounced for proximal than for distal muscles, indicating a more widespread organization of corticospinal motor projection related to proximal muscles. The combination of TMS and FSS represents a method with which functional information can be directly related to underlying cortical anatomy. This correlation will be useful in the assessment of higher brain functions with TMS.