We have developed a three-dimensional (3-D) second-order gradiometer connected to 39-channel SQUIDs for vector measurement of magnetoencephalogram (MEG) that can simultaneously detect magnetic field components perpendicular and tangential to the scalp. Each coil is orthogonally wound with Nb-Ti wire on a rectangular solid of 3×3×6 cm. To assess discrimination and separation of multiple sources, we carried out both simulation study and 3-D vector measurement of MEG with mixed auditory evoked field (AEF) and somatosensory evoked field (SEF) overlapping in time. The magnetic field distribution perpendicular to the scalp was not helpful for estimating the location and number of sources, owing to the lack of a dipole pattern, but the magnetic field distribution tangential to the scalp can provide information about new constraint conditions by visual inspection. We estimated multiple sources of mixed AEF and SEF from the MEG data of the magnetic field tangential to the scalp, and confirmed the results by comparison with superimposed source locations in MRI of a subject's head.