D- and L-amphetamine sulphate isomers, methylphenidate and atomoxetine, are effective treatments for attention-deficit hyperactivity disorder (ADHD). This study provides a detailed comparison of their effects on the synaptosomal and vesicular accumulation of dopamine (DA) and noradrenaline (NA) and release in vitro in rat prefrontal cortex and striatum. D-Amphetamine was more potent than L-amphetamine at inhibiting accumulation of DA or NA in synaptosomes and vesicles. All drugs were weaker at inhibiting the accumulation of vesicular DA and NA compared to synaptosomal accumulation and more potently inhibited NA accumulation than DA. Methylphenidate was weak at inhibiting vesicular accumulation of DA and NA compared to its potent synaptosomal effects. The D-isomer had greater potency than the L-isomer on basal and electrically stimulated striatal DA release; however the L-isomer was 2-fold more potent than the D-isomer on basal fronto-cortical NA release. The selective DA reuptake inhibitor, GBR-12909 and NA reuptake inhibitors, maprotiline and atomoxetine, had different release profiles both on the potency and magnitude of basal and stimulated DA and NA release compared to the amphetamine isomers. These results identify distinct pharmacological action by the amphetamine isomers on dopaminergic and noradrenergic neurotransmission, which may impact on their therapeutic effects in the treatment of ADHD.