In the context of virtualized-reality-based telemicromanipulation, this paper presents a visual calibration technique for an optical microscope coupled to a charge-coupled device (CCD) camera. The accuracy and flexibility of the proposed automatic virtual calibration method, based on parallel single-plane properties, are outlined. In contrast to standard approaches, a 3-D virtual calibration pattern is constructed using the micromanipulator tip with subpixel-order localization in the image frame. The proposed procedure leads to a linear systemwhose solution provides directly both the intrinsic and extrinsic parameters of the geometrical model. Computer simulations and real data have been used to test the proposed technique, and promising results have been obtained. Based on the proposed calibration techniques, a 3-D virtual microenvironment of the workspace is reconstructed through the real-time imaging of two perpendicular optical microscopes. Our method provides a flexible, easy-to-use technical alternative to the classical techniques used in micromanipulation systems.