Positioning and patterning of polystyrene particles on a silicon nitride (SiN) membrane with a microhole array has been demonstrated by positive dielectrophoresis (p-DEP). A chamber with an SiN membrane with the well-aligned microholes as the bottom substrate was positioned on an indium-tin-oxide (ITO) electrode with a 1 mm space between the bottom substrate and the ITO electrode. The chamber and the space were filled with water and a suspension of particles (10 µm diameter) in water, respectively. An AC electric signal was then applied to a microelectrode positioned at 10 µm above the SiN membrane, while the ITO electrode was connected to the ground. Particles present in the space between the SiN membrane and ITO electrode gradually moved toward the lower surface of the SiN membrane directly under the microelectrode owing to the strong electric field generated on and in localized microholes and accumulated at this position to form aggregates. For particles of 3 µm diameter, one particle was deposited in each hole (2 µm diameter) in the region directly under the microelectrode. The horizontal movement of the microelectrode gave rise to the formation of a line pattern of particles along the trail of the microelectrode because of the shift of the region with the strong electric field. These demonstrations could be applicable to arranging the particles at desired positions and in desired holes, and form particle patterns with highly flexible designs.