We have investigated spin accumulation in Ni/Au/Ni single-electron transistors assembled by atomic force microscopy. The fabrication technique is unique in that unconventional hybrid devices can be realized with unprecedented control, including real-time tunable tunnel resistances. A grid of Au disks, 30 nm in diameter and 30 nm thick, is prepared on a SiO2 surface by conventional e-beam writing. Subsequently, 30 nm thick ferromagnetic Ni source, drain, and side-gate electrodes are formed in similar process steps. The width and length of the source and drain electrodes were different to exhibit different coercive switching fields. Tunnel barriers of NiO are realized by sequential Ar and O2 plasma treatment. By use of an atomic force microscope with specially designed software, a single nonmagnetic Au nanodisk is positioned into the 25 nm gap between the source and drain electrodes. The resistance of the device is monitored in real time while the Au disk is manipulated step-by-step with angstrom-level precision. Transport measurements in magnetic field at 1.7 K reveal no clear spin accumulation in the device, which can be attributed to fast spin relaxation in the Au disk. From numerical simulations using the rate-equation approach of orthodox Coulomb blockade theory, we can put an upper bound of a few nanoseconds on the spin-relaxation time for electrons in the Au disk. To confirm the magnetic switching characteristics and spin injection efficiency of the Ni electrodes, we fabricated a test structure consisting of a Ni/NiO/Ni magnetic tunnel junction with asymmetric dimensions of the electrodes similar to those of the single-electron transistors. Magnetoresistance measurements on the test device exhibited clear signs of magnetic reversal and a maximum tunneling magnetoresistance of 10%, from which we deduced a spin polarization of about 22% in the Ni electrodes. © 2007 American Chemical Society.