Within the field of toxicology exhaled breath is used as specimen only for determination of alcohol. However, it was recently discovered that when using sensitive liquid chromatography-mass spectrometry (LC-MS) technique, amphetamine, methamphetamine, and methadone are detectable in exhaled breath following intake by drug addicts. We therefore undertook to develop a method for determination of methadone in exhaled breath condensate from patients undergoing methadonemaintenance treatment. Exhaled breath condensate was collected from 14 patients after intake of the daily methadone dose. The exhaled breath condensate was collected for 10 min using an Ecoscreen instrument. After extraction of any trapped methadone from the condensate by solid-phase extraction, the final extract was analyzed by a combined LC-MS-MS method. Recovery of methadone from breath condensate in the solid-phase extraction was 104%, no significant matrix effects were observed, and the quantification using methadone-d(3) as internal standard was accurate (10% bias) and precise (coefficient of variation 6.2%). Methadone was indisputably identified by means of the MS technique in exhaled breath condensate from all 14 patients. Identification was based on monitoring two product ions in selected reaction monitoring mode with correct relative ratio (± 20%) and correct retention time. Excretion rates ranged from 23.6 to 275 pg/min. No methadone was detected in five control subjects (< 2 pg/min). This finding confirms that methadone is present in exhaled breath from patients in methadone treatment. Collection of exhaled breath specimen is likely to be complementary to other matrices presently in use in testing for drugs-of-abuse.