BACKGROUND: Testing for antinuclear antibodies is useful for the diagnosis of systemic rheumatic diseases. Automated systems for image acquisition and interpretation of indirect immunofluorescence-based tests are increasingly used. The diagnostic performance of such automated approach in untreated patients has not been reported. METHODS: Antinuclear antibodies were measured by automated indirect immunofluorescence using Zenit G. Sight on HEp2 and HEp2000 substrate in 268 consecutive samples submitted to the laboratory for antinuclear antibody testing, and in 231 patients with a systemic rheumatic disease at the time of diagnosis, 143 blood donors, 134 patients with chronic fatigue syndrome, and 133 diseased controls. RESULTS: Image acquisition by G-Sight was of high quality. The accuracy of pattern assignment was limited. There was a significant correlation between automated estimation of fluorescence intensity (probability index of positivity) and end-point titer. Probability index interval specific likelihood ratios for systemic rheumatic disease increased with increasing level of positivity probability. With the HEp-2 substrate, the likelihood ratio for systemic lupus erythematosus was 0.06, 0.4, 6.8, 12.1, and 43.9 for a probability measure of positivity of ≤10, 11-≤30, 31-≤50, 51-≤85, and >85, respectively. CONCLUSION: Quantitative data generated by automated image acquisition facilitates standardized interpretation.