A three-dimensional position information acquiring method includes acquiring an image of a first optical image; thereafter acquiring an image of a second optical image; and performing a computation using image data of the first and second optical images. Acquisition of the image of the first optical image is based on light beams having passed through a first area. Acquisition of the image of the second optical image is based on light beams having passed through a second area. The positions of the centers of the first and second areas are both away from the optical axis of an optical system in a plane perpendicular to said optical axis. The first and second areas respectively include at least portions that do not overlap with each other. Three-dimensional position information about an observed object is acquired by the computation. The first and second areas are formed at rotationally symmetric positions. The computation performs subtraction processing using data of the first and second images, generates a subtracted image and then determined whether the observed object is displaced on the front or rear side relative to an in-focus position.