ABSTRACTIn the current experiment, we used the saccadometric test to study the effect of a single therapeutic dose of methadone on the integrity of cortico-subcortical brain functioning. In this prospective study, we used the Saccadometer System (Advanced Clinical Instrumentation, Cambridge, UK). The saccadometric test was performed before and 1.5 hours after methadone dosing. We analyzed the following saccadic parameters: latency, duration, amplitude, average and peak velocity, and processing performance (promptness) as well as a number of different types of saccades (like correct/incorrect, under/overshoot, and left-sided/right-sided). The sample consists of 40 subjects with an average 18 years of opioid addiction. The mean age is 35.3 ± 7 (80% males and 20% females). The mean period of heroin dependence is 15.3 ± 6.3 years. The mean daily dose of methadone in substitution therapy is 90 ± 26.5 mg. After administration of a single therapeutic dose of methadone, there were statistically significant differences in the values of saccade duration and latency when compared to the values before the drug administration. Average duration of saccade was significantly longer [51.40 ± 8.75 ms versus 48.93 ± 6.91 ms, z = 2.53, p = .01] and average latency was significantly longer [198.85 ± 52.57 ms versus 183.05 ± 30.95 ms, z = 2.09 p < .03]. This is the first study to test the therapeutic effect of daily methadone dosing on the integrity of the cortico-subcortical brain functions as measured by the saccadometry. More research is needed to explore the effect of illicit opioid use on the integrity of brain structures and functions, and the protective effect of opioid agonist therapy on reversing the damaging effects of illicit opioid use.