2-[(Diphenylmethyl) sulfinyl]acetamide (modafinil), prescribed principally to treat narcolepsy, is undergoing assessment for other neuropsychiatric disorders and medical conditions. The neurochemical substrates of modafinil are unresolved. We postulated that modafinil enhances wakefulness by modulating dopamine (DAT), norepinephrine (NET), or serotonin (SERT) transporter activities. In vivo, we determined DAT and NET occupancy by modafinil by positron emission tomography imaging; in vitro, we determined modafinil activity at the DAT, NET, SERT, and rhesus monkey trace amine receptor 1 (TA1). In rhesus monkey, modafinil occupancy of striatal DAT was detected by [(11)C]2beta-carbomethoxy-3beta-4-(fluorophenyl)tropane and of thalamic NET by [(11)C](S,S)-2-(alpha-(2-methoxyphenoxy)-benzyl)morpholine. In vitro, modafinil effects in DAT-human embryonic kidney (HEK), NET-HEK, and SERT-HEK cells were investigated alone or combined with the TA1 receptor. Modafinil (i.v.) occupied striatal DAT sites (5 mg/kg: 35 +/- 12%, n = 4; 8 mg/kg: 54 +/- 3%, n = 3). In thalamus, modafinil occupied NET sites (5 mg/kg: 16 +/- 7.8%, n = 6; 8 mg/kg: 44 +/- 12%; n = 2). In vitro, modafinil inhibited [(3)H]dopamine (IC(50) = 6.4 microM), [(3)H]norepinephrine (IC(50) = 35.6 microM), and [(3)H]serotonin (IC(50) > 500 microM) transport via the human DAT, NET, and SERT. Modafinil did not activate the TA1 receptor in TA1-HEK cells, but it augmented a monoamine transporter-dependent enhancement of phenethylamine activation of TA1 in TA1-DAT and TA1-NET cells, but not in TA1-SERT cells. The present data provide compelling evidence that modafinil occupies the DAT and NET in living brain of rhesus monkeys and raise the possibility that modafinil affects wakefulness by interacting with catecholamine transporters in brain.