Substitute methadone prescribing is one of the main modes of treatment for opiate dependence. This study examined the relationship between methadone dose (measured by daily dose and methadone's active (R)-enantiomer blood levels) and opiate receptor function. Nine subjects on substitute methadone (30 - 90 mg daily) received three subcutaneous injections 1.5 hours apart (saline, 5 mg and 10 mg hydromorphone, a short-acting opiate agonist) followed by measures of functional response in particular saccadic eye movements (SEMs), as well as self-report measures. Ten mg of hydromorphone significantly slowed SEM parameters (peak velocity by 15%, p < 0.005; peak acceleration by 20%, p < 0.025; peak deceleration by 26%, p < 0.025) and the SEM velocity changes correlated significantly with (R)-methadone levels (r = 0.844, p < 0.005) and with the oral dose of methadone being taken (r = 0.829, p < 0.005). Although a similar trend was observed for 5 mg, this was not significant. These finding suggest that, at higher methadone doses (resulting in higher plasma concentrations), there is significant tolerance to the action of agonists. Such studies may help in refining our understanding of the actions of methadone and the SEM measure could help in defining the degree of tolerance in individuals using street heroin.