This study confirms Leonardo's claim to have experimented on the bovine eye to determine the internal anatomy of the eye. The experiment, as described by Leonardo, was repeated in our laboratory. The study further discusses Leonardo's primary interest in the study of the eye (especially the lens), to determine how the image of an object which enters the eye in an inverted form is righted. The study shows the evolution of Leonardo's understanding of the anatomy and the physiology of vision. Initially, in keeping with his reading of the literature, the lens was placed in the centre but he made it globular. Later he promulgated two theories, reflection from the uvea and refraction within the lens to explain reversal of the image in the eye. Subsequently he rejected the first theory and, putting credence in the second theory, experimented (1509) to show that the lens is globular and is centrally placed. The fact that the present knowledge about the lens is at variance from his findings is not because he did not carry out the experiment, as suggested by some modern authors, but because of the limitation of the techniques available to him at the time.