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In fundus photography, the task of fine focusing the image is demanding and lack of focus is quite often the cause of suboptimal photographs. The introduction of digital cameras has provided an opportunity to automate the task of focusing. We have developed a software algorithm capable of identifying best focus. The auto-focus (AF) method is based on an algorithm we developed to assess the sharpness of an image. The AF algorithm was tested in the prototype of a semi-automated nonmydriatic fundus camera designed to screen in the primary care environment for major eye diseases. A series of images was acquired in volunteers while focusing the camera on the fundus. The image with the best focus was determined by the AF algorithm and compared to the assessment of two masked readers. A set of fundus images was obtained in 26 eyes of 20 normal subjects and 42 eyes of 28 glaucoma patients. The 95% limits of agreement between the readers and the AF algorithm were -2.56 to 2.93 and -3.7 to 3.84 diopter and the bias was 0.09 and 0.71 diopter, for the two readers respectively. On average, the readers agreed with the AF algorithm on the best correction within less than 3/4 diopter. The intraobserver repeatability was 0.94 and 1.87 diopter, for the two readers respectively, indicating that the limit of agreement with the AF algorithm was determined predominantly by the repeatability of each reader. An auto-focus algorithm for digital fundus photography can identify the best focus reliably and objectively. It may improve the quality of fundus images by easing the task of the photographer.