The aim of this study was to elucidate whether autonomic nervous system dysfunction exists in patients with Behçet's disease by pupillometric tests. Thirty-one patients with Behçet's disease with a mean age of 41.3 years (range 21-64) and 41 control subjects with a mean age of 39.5 years (range 18-66) were selected for the study. To test the autonomic nervous system, four pupillometric techniques were used: pupil cycle time (PCT), dark-adapted pupil size (DAPS), 0.05% pilocarpine drop test, and 1% phenylephrine drop test. In all four tests, there were significant differences between the patients and controls. Mean PCTs were 1,156 ms (range 856-1,560 ms) and 919 ms (range 650-1,261 ms) in the patients and controls, respectively (p < 0.0001). The mean DAPS was 0.45 (range 0.31-0.66) in the patients, whereas it was 0.56 (range 0.42-0.67) in controls (p < 0.001). Iris sensitivity to both 0.05% pilocarpine and 1% phenylephrine showed significant differences between patients and controls, respectively (p < 0.05, p < 0.05). Among all four tests, only 0.05% pilocarpine sensitivity was correlated with the duration of Behçet's disease (p < 0.05). The results suggest that the autonomic nervous system innervating the iris is affected in Behçet's disease. This involvement may be due to the vasculitic nature of Behçet's disease.