A tracking algorithm has been developed to efficiently track a moving object in two-dimensional image space. The algorithm employs a limited exhaustive template matching scheme that combines the accuracy of an exhaustive search with the computational efficiency of a coarse-fine template matching scheme. The overall result is an accurate, time-efficient tracking algorithm. After providing a theoretical discussion of the algorithm, three separate biomedical applications of the algorithm are described: (1) stabilizing an irradiating laser on the retinal surface for photocoagulation treatment, (2) measuring target fixation eye movement to construct pattern densities at the retina, and (3) tracking a rat swimming in a Morris Water Maze for psychophysiological studies. Results for each application is provided. The paper concludes with a discussion of the relative merits of the tracking algorithm and recommendations for methods to improve the performance of the algorithm. © 2001 SPIE and IS&T.