The Heidelberg Retina Tomograph is a confocal scanning laser ophthalmoscope which obtains three-dimensional images of the optic nerve head and the retina in the human eye. Because of its potential investigative uses in experimental animal models of glaucoma, we examined its variability with optic nerve head measurements in the rabbit eye. Three topographic images, recorded on different days, were acquired from the right eye of 5 New Zealand white rabbits over 3 weeks. To estimate the lowest possible variability, a second series of three images was recorded in a single setting without displacement of the rabbits. The average coefficient of variability (standard deviation/mean) for the estimate of the optic nerve head cup volume (volume below surface) was 11.1% in the independent series and was decreased to 3.0% in the sequentially recorded series without displacement of the rabbits (P < 0.001). These values indicate a comparable variability for the estimate of the optic nerve head cup volume in the rabbit compared with those reported for the human eye. This variability is considerably decreased by maximally standardizing the image acquisition position, suggesting that variability largely depends on the alignment between the subject and the laser-scanner.