PURPOSE. To perform ocular motility recordings of infantile nystagmus (IN) in RPE65-deficient canines and determine whether they can be used as a motor indicator of restored retinal function to investigate the effects of gene therapy. METHODS. Treated and untreated canines were comfortably suspended in a custom-built sling and encouraged to fixate on distant targets at gaze angles varying between ±15° horizontally and ±10° vertically. Ocular motility recordings were made, using two distinct methods - infrared reflection and high-speed video. The resultant recordings from three untreated, four treated, and three pre- and post-treatment dogs were analyzed for using the eXpanded Nystagmus Acuity Function (NAFX), which yields an objective assessment of best potential visual acuity, based on the duration and repeatable accuracy of foveation and centralisation. RESULTS. During fixation, the untreated dogs exhibited large-amplitude, classic IN waveforms, including pendular and jerk in both the horizontal and vertical planes, which prevented them from keeping the targets within the area centralis (the region of highest receptor density, spanning ±3° horizontally by ±1.5° vertically, analogous to the fovea). Some untreated dogs also had small-amplitude (0.5-1°), high-frequency (6-9 Hz) oscillations. Under the same conditions, successfully treated canines no longer exhibited clinically detectable IN. Their IN was converted to waveforms with very low amplitudes that yielded higher NAFX values and allowed target images to remain well within the area centralis. Of note, uniocular treatment appeared to damp the IN in both eyes. Behaviorally, the treated dogs were able to successfully navigate through obstacles more easily without inadvertent contact, a task beyond the untreated dogs' ability. CONCLUSIONS. Gene therapy that successfully restored retinal function also reduced the accompanying IN to such a great extent that it was not clinically detectable ∼90% of the time in many of the dogs. IN improvement, as quantified by the NAFX, is an objective motor indicator of visual improvement due to gene therapy. Copyright © Association for Research in Vision and Ophthalmology.